Calcium independent cytosolic phospholipase A2/B enzymes

ABSTRACT

The invention provides a novel calcium-independent cytosolic phospholipase A 2 /B enzyme, polynucleotides encoding such enzyme and methods for screening unknown compounds for anti-inflammatory activity mediated by the arachidonic acid cascade.

[0001] This application is a continuation-in-part of application Ser.No. 08/281,193, filed Jul. 27, 1994.

[0002] The present invention relates to a purified calcium independentcytosolic phospholipase A₂/B enzymes which are useful for assayingchemical agents for anti-inflammatory activity.

BACKGROUND OF THE INVENTION

[0003] The phospholipase A₂ enzymes comprise a widely distributed familyof enzymes which catalyze the hydrolysis of the acyl ester bond ofglycerophospholipids at the sn-2 position. One kind of phospholipase A₂enzymes, secreted phospholipase A₂ or sPLA₂, are involved in a number ofbiological functions, including phospholipid digestion, the toxicactivities of numerous venoms, and potential antibacterial activities. Asecond kind of phospholipase A₂ enzymes, the intracellular phospholipaseA₂ enzymes, also known as cytosolic phospholipase A₂ or cPLA₂, areactive in membrane phospholipid turnover and in regulation ofintracellular signalling mediated by the multiple components of thewell-known arachidonic acid cascade. One or more cPLA₂ enzymes arebelieved to be responsible for the rate limiting step in the arachidonicacid cascade, namely, release of arachidonic acid from membraneglycerophospholipids. The action of cPLA₂ also results in biosynthesisof platelet activating factor (PAF).

[0004] The phospholipase B enzymes are a family of enzymes whichcatalyze the hydrolysis of the acyl ester bond of glycerophospholipidsat the sn-1 and sn-2 positions. The mechanism of hydrolysis is unclearbut may consist of initial hydrolysis of the sn-2 fatty acid followed byrapid cleavage of the sn-1 substituent, i.e., functionally equivalent tothe combination of phospholipase A₂ and lysophospholipase (Saito et al.,Methods of Enzymol., 1991, 197, 446; Gassama-Diagne et al., J. Biol.Chem., 1989, 264, 9470). Whether these two events occur at the same ortwo distinct active sites has not been resolved. It is also unknown ifthese enzymes have a preference for the removal of unsaturated fattyacids, in particular arachidonic acid, at the sn-2 position andaccordingly contribute to the arachidonic acid cascade.

[0005] Upon release from the membrane, arachidonic acid may bemetabolized via the cyclooxygenase pathway to produce the variousprostaglandins and thromboxanes, or via the lipoxygenase pathway toproduce the various leukotrienes and related compounds. Theprostaglandins, leukotrienes and platelet activating factor are wellknown mediators of various inflammatory states, and numerousanti-inflammatory drugs have been developed which function by inhibitingone or more steps in the arachidonic acid cascade. Use of the presentanti-inflammatory drugs which act through inhibition of arachidonic acidcascade steps has been limited by the existence of side effects whichmay be harmful to various individuals.

[0006] A very large industrial effort has been made to identifyadditional anti-inflammatory drugs which inhibit the arachidonic acidcascade. In general, this industrial effort has employed the secretedphospholipase A₂ enzymes in inhibitor screening assays, for example, asdisclosed in U.S. Pat. No. 4,917,826. However, because the secretedphospholipase A₂ enzymes are extracellular proteins (i.e., notcytosolic) and are not specific for hydrolysis of arachidonic acid, theyare presently not believed to participate directly in the arachidonicacid cascade. While some inhibitors of the small secreted phospholipaseA₂ enzymes have anti-inflammatory action, such as indomethacin,bromphenacyl bromide, mepacrine, and certain butyrophenones as disclosedin U.S. Pat. No. 4,239,780, it is presently believed that inhibitorscreening assays should employ cytosolic phospholipase A₂ enzymes whichdirectly participate in the arachidonic acid cascade.

[0007] An improvement in the search for anti-inflammatory drugs whichinhibit the arachidonic acid cascade was developed in commonly assignedU.S. Pat. No. 5,322,776, incorporated herein by reference. In thatapplication, a cytosolic form of phospholipase A₂ was identified,isolated, and cloned. Use of the cytosolic form of phospholipase A₂ toscreen for anti-inflammatory drugs provides a significant improvement inidentifying inhibitors of the arachidonic acid cascade. The cytosolicphospholipase A₂ disclosed in U.S. Pat. No. 5,322,776 is a 110 kDprotein which depends on the presence of elevated levels of calciuminside the cell for its activity. The cPLA₂ of U.S. Pat. No. 5,322,776plays a pivotal role in the production of leukotrienes andprostaglandins initiated by the action of pro-inflammatory cytokines andcalcium mobilizing agents. The cPLA₂ of U.S. Pat. No. 5,322,776 isactivated by phosphorylation on serine residues and increasing levels ofintracellular calcium, resulting in translocation of the enzyme from thecytosol to the membrane where arachidonic acid is selectively hydrolyzedfrom membrane phospholipids.

[0008] In addition to the cPLA₂ of U.S. Pat. No. 5,322,776, some cellscontain calcium independent phospholipase A₂/B enzymes. For example,such enzymes have been identified in rat, rabbit, canine and human-hearttissue (Gross, TCM, 1991, 2, 115; Zupan et al., J. Med. Chem., 1993, 36,95; Hazen et al., J. Clin. Invest., 1993, 91, 2513; Lehman et al., J.Biol. Chem., 1993, 268, 20713; Zupan et al., J. Biol. Chem., 1992, 267,8707; Hazen et al., J. Biol. Chem., 1991, 266, 14526; Loeb et al., J.Biol. Chem., 1986, 261, 10467; Wolf et al., J. Biol. Chem., 1985, 260,7295; Hazen et al., Meth. Enzymol., 1991, 197, 400; Hazen et al., J.Biol. Chem., 1990, 265, 10622; Hazen et al., J. Biol. Chem., 1993, 268,9892; Ford et al., J. Clin. Invest., 1991, 88, 331; Hazen et al., J.Biol. Chem., 1991, 266, 5629; Hazen et al., Circulation Res., 1992, 70,486; Hazen et al., J. Biol. Chem., 1991, 266, 7227; Zupan et al., FEBS,1991, 284, 27), as well as rat and human pancreatic islet cells(Ramanadham et al., Biochemistry, 1993, 32, 337; Gross et al.,Biochemistry, 1993, 32, 327), in the macrophage-like cell line, P388D₁(Ulevitch et al., J. Biol. Chem., 1988, 263, 3079; Ackermann et al., J.Biol. Chem., 1994, 269, 9227; Ross et al., Arch. Biochem. Biophys.,1985, 238, 247; Ackermann et al., FASEB Journal, 1993, 7(7), 1237), invarious rat tissue cytosols (Nijssen et al., Biochim. Biophys. Acta,1986, 876, 611; Pierik et al., Biochim. Biophys. Acta, 1988, 962, 345;Aarsman et al., J. Biol. Chem., 1989, 264, 10008), bovine brain (Ueda etal., Biochem. Biophys, Res. Comm., 1993, 195, 1272; Hirashima et al., J.Neurochem., 1992, 59, 708), in yeast (Saccharomyces cerevisiae)mitochondria (Yost et al., Biochem. International, 1991, 24, 199),hamster heart cytosol (Cao et al., J. Biol. Chem., 1987, 262, 16027),rabbit lung microsomes (Angle et al., Biochim. Biophys. Acta, 1988, 962,234) and guinea pig intestinal brush-border membrane (Gassama-Diagne etal., J. Biol. Chem., 1989, 264, 9470).

[0009] It is believed that the calcium independent phospholipase A₂/Benzymes may perform important functions in release of arachidonic acidin specific tissues which are characterized by unique membranephospholipids, by generating lysophospholipid species which aredeleterious to membrane integrity or by remodeling of unsaturatedspecies of membrane phospholipids through deacylation/reacylationmechanisms. The activity of such a phospholipase may well be regulatedby mechanisms that are different from that of the cPLA₂ of U.S. Pat. No.5,322,776. In addition the activity may be more predominant in certaininflamed tissues over others. Although the enzymatic activity is notdependent on calcium this does not preclude a requirement for calcium invivo, where the activity may be regulated by the interaction of otherprotein(s) whose function is dependent upon a calcium flux.

SUMMARY OF THE INVENTION

[0010] In certain embodiments, the present invention providescompositions comprising a purified phospholipase enzyme characterized by(a) activity in the absence of calcium; (b) a molecular weight of 86 kDon SDS-PAGE; and (c) the presence of one or more amino acid sequencesselected from the group consisting of NPHSGFR (SEQ ID NO:3), XASXGLNQVNK(SEQ ID NO:4) (X is preferably N or A), YGASPLHXAK (SEQ ID NO:5) (X ispreferably W), DNMEMIK (SEQ ID NO:6), GVYFR (SEQ ID NO:7), MKDEVFR (SEQID NO:8), EFGEHTK (SEQ ID NO:9), VMLTGTLSDR (SEQ ID NO:10), XYDAPEVIR(SEQ ID NO:11) (X is preferably N), FNQNINLKPPTQPA (SEQ ID NO:12),XXGAAPTYFRP (SEQ ID NO:13) (X is preferably S), TVFGAK (SEQ ID NO:14),and XWSEMVGIQYFR (SEQ ID NO:15) (X is preferably A), wherein Xrepresents any amino acid residue.

[0011] In other embodiments, the invention provides compositionscomprising a purified phospholipase enzyme characterized by (a) activityin the absence of calcium; (b) a molecular weight of 86 kD on SDS-PAGE;and (c) the presence of one or more amino acid sequences selected fromthe group consisting of YGASPLHXAK, MKDEVFR, EFGEHTK, VMLTGTLSDR,XXGAAPTYFRP and TVFGAK, wherein X represents any amino acid residue.

[0012] Certain embodiments provide compositions comprising a purifiedmammalian calcium independent phospholipase A₂/B enzyme.

[0013] In other embodiments, the enzyme is further characterized byactivity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine (preferably aspecific activity of about 1 μmol to about 20 μmol per minute permilligram, more preferably a specific activity of about 1 μmol to about5 μmol per minute per milligram); by a pH optimum of 6; and/or by theabsence of stimulation by adenosine triphosphate in the liposome assay.

[0014] In other embodiments, the invention provides isolatedpolynucleotides comprising a nucleotide sequence selected from the groupconsisting of: (a) the nucleotide sequence of SEQ ID NO:1; (b) anucleotide sequence encoding the amino acid sequence of SEQ ID NO:2; (c)a nucleotide sequence encoding a fragment of the amino acid sequence ofSEQ ID NO:2 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (d) a nucleotidesequence capable of hybridizing with the sequence of (a), (b) or (c)which encodes a peptide having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; and (e) allelicvariants of the sequence of (a). Other embodiments provide an isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of: (a) the nucleotide sequence of SEQ ID NO:16; (b) anucleotide sequence encoding the amino acid sequence of SEQ ID NO:17;(c) a nucleotide sequence encoding a fragment of the amino acid sequenceof SEQ ID NO:17 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (d) the nucleotidesequence of SEQ ID NO:18; (e) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:19; (f) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO:19 having activity in amixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (g) the nucleotidesequence of SEQ ID NO:20; (h) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:21; (i) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO:21 having activity in amixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (j) the nucleotidesequence of SEQ ID NO:22; (k) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:23; (l) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO:23 having activity in amixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (m) a nucleotidesequence capable of hybridizing with the sequence of any of (a)-(l)which encodes a peptide having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; and (n) allelicvariants of the sequence of (a), (d), (g) or (j). Expression vectorscomprising such polynucleotides and host cells transformed with suchvectors are also provided by the present invention. Compositionscomprising peptides encoded by such polynucleotides are also provided.

[0015] The present invention also provides processes for producing aphospholipase enzyme, said process comprising: (a) establishing aculture of the host cell transformed with a cPLA₂/B encodingpolynucleotide in a suitable culture medium; and (b) isolating saidenzyme from said culture. Compositions comprising a peptide madeaccording to such processes are also provided.

[0016] Certain embodiments of the present invention provide compositionscomprising a peptide comprising an amino acid sequence selected from thegroup consisting of: (a) the amino acid sequence of SEQ ID NO:2; and (b)a fragment of the amino acid sequence of SEQ ID NO:2 having activity ina mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.

[0017] Other embodiments provide compositions comprising a peptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:17; (b) a fragment of the aminoacid sequence of SEQ ID NO:17 having activity in a mixed micelle assaywith 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (c) the aminoacid sequence of SEQ ID NO:19; (d) a fragment of the amino acid sequenceof SEQ ID NO:19 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (e) the amino acidsequence of SEQ ID NO:21; (f) a fragment of the amino acid sequence ofSEQ ID NO:21 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (g) the amino acidsequence of SEQ ID NO:23; and (h) a fragment of the amino acid sequenceof SEQ ID NO:23 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.

[0018] The present invention also provides methods for identifying aninhibitor of phospholipase activity, said method comprising: (a)combining a phospholipid, a candidate inhibitor compound, and acomposition comprising a phospholipase enzyme peptide; and (b) observingwhether said phospholipase enzyme peptide cleaves said phospholipid andreleases fatty acid thereby, wherein the peptide composition is one ofthose described above. Inhibitor of phospholipase activity identified bysuch methods, pharmaceutical compositions comprising a therapeuticallyeffective amount of such inhibitors and a pharmaceutically acceptablecarrier, and methods of reducing inflammation by administering suchpharmaceutical compositions to a mammalian subject are also provided.

[0019] Polyclonal and monoclonal antibodies to the peptides of theinvention are also provided.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1: Fractions containing activity eluted from a Mono P columnwere examined by reducing SDS-PAGE on a 4-20% gradient gel. Activity ofeach fraction is show above the gel and the 86 kD band is indicated onthe silver stained gel. Molecular weight markers are indicated.

[0021]FIG. 2: Active fractions from a Mono p/Heparin column werecombined and further purified on a size exclusion column. Activityeluted in the 250-350 kD size range. Examination of the fractions bySDS-PAGE under reducing conditions on 4-20% gel indicated only oneprotein band correlated with activity at 86 kD. Molecular weight markersare indicated.

[0022]FIG. 3: Active fractions from Mono P eluate and cPLA₂ (0.1-1.0 μg)were analyzed on two 4-20% SDS gels under reducing conditions run inparallel. One gel was silver stained (A) and in the other gel theproteins were transferred to nitrocellulose. the blot was than probedwith an anti-cPLA₂ polyclonal antibody and reactive proteins werevisualized with the ECL system (Amersham) (B). Molecular weight markersare indicated.

[0023]FIG. 4: The activity of the calcium-independent phospholipaseeluted from a Mono P/Heparin column and cPLA₂ were compared underconditions which favor each enzyme; pH 7, 10% glycerol in the absence ofcalcium and pH 9, 70% glycerol in the presence of calcium, respectively.

[0024]FIG. 5: Activity in the cytosolic extracts of COS cellstransfected with: no DNA; plasmid (pED) containing no inserted gene;clone 9 in the antisense orientation; and clones 49, 31 and 9 expressedin pED. The extracts were analyzed under two different assay conditionsdescribed for the data presented in FIG. 4.

[0025]FIG. 6: A comparison of sn-2 fatty acid hydrolysis by activityeluted from a Mono P/Heparin column as a function of the fatty acidsubstituent at either the sn-1 or sn-2 position and the head group.HAPC, SAPC, PLPC, POPC, PPPC, LYSO and PAPC indicate1-hexadecyl-2-arachidonyl-, 1-stearoyl-2-arachidonyl-,1-palmitoyl-2-linoleyl-, 1-palmitoyl-2-oleyl-, 1-palmitoyl-2-palmitoyl-,1-palmitoyl-, 1-palmitoyl-2-arachidonyl-phosphatidylcholine,respectively. PAPE and SAPI indicate1-palmitoyl-2-arachidonyl-phosphotidylethanolamine and1-stearoyl-2-arachidonyl-phosphoinositol, respectively. In all cases the¹⁴C-labelled fatty acid is in the sn-2 position.

[0026]FIG. 7: A 4-20% SDS-PAGE of lysates (5×10¹⁰ cpm/lane) of35S-methionine labelled COS cells transfected with, no DNA, pED (noinsert), clone 9 reverse orientation, clones 9, 31 and 49; lanes 1-6,respectively. Molecular weight markers are indicated.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present inventors have found surprisingly a calciumindependent cytosolic phospholipase enzyme, designated calciumindependent cytosolic phospholipase A₂/B or calcium independent cPLA₂/B,purified from the cytosol of Chinese hamster ovary (CHO) cells. Theactivity was also present in the cytosol of tissues and cell extractslisted in Table I. TABLE I mixed micelle pH 7 liposome pH 7 tissue/cell(pmol/min/mg) (pmol/min/mg) rat brain 1-2 rat heart 0.3-0.5 bovine brain0.4 pig heart 0.8 CHO-Dukx 10-20 2-5 U937 (ATCC CRL1593) 2 FBHE (ATCCCRL1395) 2 H9c2 (ATCC Ccl 108) 15

[0028] The enzyme was originally purified by more than 8,000-fold fromCHO cells by sequential chromatography on diethylaminoethane (DEAE),phenyl and heparin-toyopearl, followed by chromatofocussing on Mono P(as described further in Example 1). In addition the activity could befurther purified by size exclusion chromatography after the Mono Pcolumn. The enzyme eluted from the size exclusion chromatography columnin the 250-350 kD range, indicating the active enzyme may consist of amultimeric complex, or may possibly be associated with phospholipids.

[0029] The calcium independent phospholipase activity correlated with asingle major protein band of 86 kD on denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of active fractions fromthe Mono P and size exclusion chromatographic steps; in the latter noprotein bands were observed in the 250-350 kD range. The specificactivity of the enzyme is about 1 μmol to about 20 μmol per minute permilligram based on the abundance of the 86 kD band in the most activefractions eluted from the Mono P and size exclusion columns in the mixedmicelle assay (Example 3B). The protein band was not recognized by apolyclonal antibody directed against the calcium dependent cPLA₂ of U.S.Pat. No. 5,322,776.

[0030] The calcium independent phospholipase of the present inventionhas a pH optimum of 6; its activity is suppressed by calcium (in allassays) and by triton X-100 (in the assay of Example 3A); and is notstimulated by adenosine triphosphate (ATP) (in the assay of Example 3A).The enzyme is inactivated by high concentration denaturants, e.g. ureaabove 3M, and by detergents, e.g. CHAPS and octyl glucoside. Thecalcium-independent phospholipase favors hydrolysis by several fold ofunsaturated fatty acids, e.g. linoleyl, oleyl and arachidonyl, at thesn-2 position of a phospholipid compared with palmitoyl. In additionthere is a preference for palmitoyl at the sn-1 position over hexadecylor stearoyl for arachidonyl hydrolysis at the sn-2 position. In terms ofhead group substituents there is a clear preference for inositol overcholine or ethanolamine when arachidonyl is being hydrolyzed at the sn-2position. Further, as with cPLA₂ of U.S. Pat. No. 5,322,776, there is asignificant lysophospholipase activity, i.e. hydrolysis of palmitoyl atthe sn-1 position when there is no fatty acid substituent at the sn-2position. Finally, hydrolysis of fatty acid substituents in the sn-1 orsn-2 in PAPC were compared where either palmitoyl or arachidonyl werelabelled with ¹⁴C. Fatty acids were removed at both positions with thesn-2 position having a higher initial rate of hydrolysis by 2-3 fold.This result may indicate sequential hydrolysis of the arachidonylsubstituent followed by rapid cleavage of palmitoyl in thelysophospholipid species, which is suggested by the hydrolysis of theindividual lipid species. The similar rates of hydrolysis of fatty acidsubstituents at the sn-1 (palmitoyl) or sn-2 (arachidonyl) positions,where the radioactive label is in either position, is indicative of aphospholipase B activity. However, the fatty acid substituent at thesn-2 position clearly influences the PLB activity, not the sn-1 fattyacid, since hydrolysis of 1,2-dipalmitoyl substituted phospholipids issubstantially less than for the 1-palmitoyl-2-arachidonyl species. Theseresults can be clarified by studying the hydrolysis rates at eachposition of isotopically dual labelled phospholipids, e.g. ³H and ¹⁴Ccontaining fatty acids at the sn-1 and sn-2 positions, respectively.Therefore, it is prudent to designate the enzyme as a phospholipaseA₂/B.

[0031] A cDNA encoding the calcium independent cPLA₂/B of the presentinvention was isolated as described in Example 4. The sequence of thecDNA is reported as SEQ ID NO:1. The amino acid sequence encoded by suchcDNA is SEQ ID NO:2. The invention also encompasses allelic variationsof the cDNA sequence as set forth in SEQ ID NO:1, that is,naturally-occurring alternative forms of the cDNA of SEQ ID NO: 1 whichalso encode phospholipase enzymes of the present invention.

[0032] Other cDNAs encoding a calcium independent cPLA₂/B of the presentinvention were isolated from human cDNA sources. Two clones identifiedas “19a” and “19b” were isolated from a Raij cell DNA library derivedfrom Burkitt's lymphoma (ATCC CCL86, commercially available fromClonetech) using a probe derived from the CHO sequence (a 2.1 kbSalI-SmaI fragment). Clones 19a and 19b were deposited with the AmericanType Culture Collection on Nov. 7, 1995 as accession numbers ATCC______and ATCC______. The nucleotide sequences of clones 19a and 19b arereported in SEQ ID NO:16 and SEQ ID NO:18, respectively. SEQ ID NO:17and SEQ ID NO:18 report the corresponding amino acid sequences encodedby the coding regions of clones 19a and 19b, respectively. Clones 19aand 19b are both partial clones of the full-length human enzyme.

[0033] SEQ ID NO:20 and SEQ ID NO:22 report the nucleotide seqeunces ofalternative ways in which clones 19a and 19b can be spliced to encode alonger partial clone for the full-length human enzyme. The splice occursafter nucleotide 1225 in SEQ ID NO:20 and after nucleotide 1228 in SEQID NO:22. The corresponding spliced amino acid sequences are reported inSEQ ID NO:21 and SEQ ID NO:23. Spliced cDNA clones can be made fromclones 19a and 19b in accordance with methods known to those skilled inthe art.

[0034] Full-length clones encoding the human enzyme can be isolated byprobing human cDNA libraries containing full-length clones using probesderived from SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.

[0035] Also included in the invention are isolated DNAs which hybridizeto the DNA sequence set forth in SEQ ID NO:1, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20 or SEQ ID NO:22 under stringent (e.g. 4×SSC at 65°C. or 50% formamide and 4×SSC at 42° C.), or relaxed (4×SSC at 50° C. or30-40% formamide at 42° C.) conditions.

[0036] The isolated polynucleotides of the invention may be operablylinked to an expression control sequence such as the pMT2 or pEDexpression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19,4485-4490 (1991), in order to produce the phospholipase enzyme peptidesrecombinantly. Many suitable expression control sequences are known inthe art. General methods of expressing recombinant proteins are alsoknown and are exemplified in R. Kaufman, Methods in Enzymology 185,537-566 (1990). As defined herein “operably linked” means enzymaticallyor chemically ligated to form a covalent bond between the isolatedpolynucleotide of the invention and the expression control sequence, insuch a way that the phospholipase enzyme peptide is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

[0037] A number of types of cells may act as suitable host cells forexpression of the phospholipase enzyme peptide. Suitable host cells arecapable of attaching carbohydrate side chains characteristic offunctional phospholipase enzyme peptide. Such capability may arise byvirtue of the presence of a suitable glycosylating enzyme within thehost cell, whether naturally occurring, induced by chemical mutagenesis,or through transfection of the host cell with a suitable expressionplasmid containing a polynucleotide encoding the glycosylating enzyme.Host cells include, for example, monkey COS cells, Chinese Hamster Ovary(CHO) cells, human kidney 293 cells, human epidermal A431 cells, humanColo205 cells, 3T3 cells, CV-1 cells, other transformed primate celllines, normal diploid cells, cell strains derived from in vitro cultureof primary tissue, primary explants, HeLa cells, mouse L cells, BHK,HL-60, U937, or HaK cells.

[0038] The phospholipase enzyme peptide may also be produced by operablylinking the isolated polynucleotide of the invention to suitable controlsequences in one or more insect expression vectors, and employing aninsect expression system. Materials and methods for baculovirus/insectcell expression systems are commercially available in kit form from,e.g., Invitrogen, San Diego, Calif. U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference.

[0039] Alternatively, it may be possible to produce the phospholipaseenzyme peptide in lower eukaryotes such as yeast or in prokaryotes suchas bacteria. Potentially suitable yeast strains include Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida,or any yeast strain capable of expressing heterologous proteins.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the phospholipase enzymepeptide is made in yeast or bacteria, it is necessary to attach theappropriate carbohydrates to the appropriate sites on the protein moietycovalently, in order to obtain the glycosylated phospholipase enzymepeptide. Such covalent attachments may be accomplished using knownchemical or enzymatic methods.

[0040] The phospholipase enzyme peptide of the invention may also beexpressed as a product of transgenic animals, e.g., as a component ofthe milk of transgenic cows, goats, pigs, or sheep which arecharacterized by somatic or germ cells containing a polynucleotideencoding the phospholipase enzyme peptide.

[0041] The phospholipase enzyme peptide of the invention may be preparedby culturing transformed host cells under culture conditions necessaryto express a phospholipase enzyme peptide of the present invention. Theresulting expressed protein may then be purified from culture medium orcell extracts as described in the examples below.

[0042] Alternatively, the phospholipase enzyme peptide of the inventionis concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. Sulfopropylgroups are preferred (e.g., S-Sepharose® columns). The purification ofthe phospholipase enzyme peptide from culture supernatant may alsoinclude one or more column steps over such affinity resins asconcanavalin A-agarose, heparin-toyopear® or Cibacrom blue 3GASepharose®; or by hydrophobic interaction chromatography using suchresins as phenyl ether, butyl ether, or propyl ether; or byimmunoaffinity chromatography.

[0043] Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the phospholipase enzyme peptide. Some or allof the foregoing purification steps, in various combinations, can alsobe employed to provide a substantially homogeneous isolated recombinantprotein. The phospholipase enzyme peptide thus purified is substantiallyfree of other mammalian proteins and is defined in accordance with thepresent invention as “isolated phospholipase enzyme peptide”.

[0044] The calcium independent cPLA₂/B of the present invention isdistinct from the cPLA₂ of U.S. Pat. No. 5,322,776 and frompreviously-described calcium independent phospholipase A₂ enzymes (suchas those described by Gross et al., supra; and Ackermann et al., supra).The enzyme of the present invention differs from the cPLA₂ of the ′776patent in the following ways:

[0045] (1) its activity is not calcium dependent;

[0046] (2) it is more active in 10% glycerol than in 70% glycerol;

[0047] (3) it has a molecular weight of 86 kD, not 110 kD as for cPLA₂;

[0048] (4) it has a pH optimum of 6, not greater than 8 as for cPLA₂;

[0049] (5) it hydrolyzes fatty acids at sn-1 as well as sn-2;

[0050] (6) it binds to heparin, while cPLA₂ does not;

[0051] (7) it elutes from an anion exchange column at 0.1-0.2 M NaCl,while cPLA₂ elutes at 0.3-0.4 M NaCl; and

[0052] (8) it does not bind to anti-cPLA₂ polyclonal antibody.

[0053] The enzyme of the present invention differs from the calciumindependent enzyme of Gross et al. in the following characteristics:

[0054] (1) it has a molecular weight of 86 kD, not 40 kD as for theGross enzyme;

[0055] (2) it is not homologous at the protein level to rabbit skeletalmuscle phosphofructokinase in contrast to the 85 kD putative regulatoryprotein associated with the 40 kD Gross enzyme;

[0056] (3) hydrolysis at the sn-2 position is favored by an acyl-linkedfatty acid at the sn-1 position in contrast to ether-linked fatty acidswith the Gross enzyme;

[0057] (4) its does not bind to an ATP column and was not activated byATP in a liposome assay compared to the Gross enzyme; and

[0058] (5) it was active in a mixed micelle assay containing TritonX-100.

[0059] The enzyme of the present invention differs from the calciumindependent enzyme of Ackermann et al. (the “Dennis enzyme”)in thefollowing characteristics:

[0060] (1) it does not bind to an ATP column;

[0061] (2) it binds to an anion exchange column (mono Q), while theDennis enzyme remains in the unbound fraction;

[0062] (3) it has a molecular weight of 86 kD, not 74 kD as for theDennis enzyme;

[0063] (4) it has substantial lysophospholipase activity and isrelatively inactive on phospholipids containing ether-linked fatty acidsat the sn-1 position in a liposome assay; and

[0064] (5) it appears to hydrolyze fatty acid substituents at the sn-1and sn-2 positions of a phospholipid, whereas the Dennis enzyme favorshydrolysis at the sn-2 position.

[0065] The calcium independent cPLA₂/B of the present invention may beused to screen unknown compounds having anti-inflammatory activitymediated by the various components of the arachidonic acid cascade. Manyassays for phospholipase activity are known and may be used with thecalcium independent phospholipase A₂/B on the present invention toscreen unknown compounds. For example, such an assay may be a mixedmicelle assay as described in Example 3. Other known phospholipaseactivity assays include, without limitation, those disclosed in U.S.Pat. No. 5,322,776. These assays may be performed manually or may beautomated or robotized for faster screening. Methods of automation androbotization are known to those skilled in the art.

[0066] In one possible screening assay, a first mixture is formed bycombining a phospholipase enzyme peptide of the present invention with aphospholipid cleavable by such peptide, and the amount of hydrolysis inthe first mixture (B₀) is measured. A second mixture is also formed bycombining the peptide,' the phospholipid and the compound or agent to bescreened, and the amount of hydrolysis in the second mixture (B) ismeasured. The amounts of hydrolysis in the first and second mixtures arecompared, for example, by performing a B/B₀ calculation. A compound oragent is considered to be capable of inhibiting phospholipase activity(i.e., providing anti-inflammatory activity) if a decrease in hydrolysisin the second mixture as compared to the first mixture is observed. Theformulation and optimization of mixtures is within the level of skill inthe art, such mixtures may also contain buffers and salts necessary toenhance or to optimize the assay, and additional control assays may beincluded in the screening assay of the invention.

[0067] Other uses for the calcium independent cPLA₂/B of the presentinvention are in the development of monoclonal and polyclonalantibodies. Such antibodies may be generated by employing purified formsof the calcium independent cPLA₂ or immunogenic fragments thereof as anantigen using standard methods for the development of polyclonal andmonoclonal antibodies as are known to those skilled in the art. Suchpolyclonal or monoclonal antibodies are useful as research or diagnostictools, and further may be used to study phospholipase A₂ activity andinflammatory conditions.

[0068] Pharmaceutical compositions containing anti-inflammatory agents(i.e., inhibitors) identified by the screening method of the presentinvention may be employed to treat, for example, a number ofinflammatory conditions such as rheumatoid arthritis, psoriasis, asthma,inflammatory bowel disease and other diseases mediated by increasedlevels of prostaglandins, leukotriene, or platelet activating factor.Pharmaceutical compositions of the invention comprise a therapeuticallyeffective amount of a calcium independent cPLA₂ inhibitor compound firstidentified according to the present invention in a mixture with anoptional pharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The term “therapeutically effective amount” means the total amount ofeach active component of the method or composition that is sufficient toshow a meaningful patient benefit, i.e., healing or amelioration ofchronic conditions or increase in rate of healing or amelioration. Whenapplied to an individual active ingredient, administered alone, the termrefers to that ingredient alone. When applied to a combination, the termrefers to combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. A therapeutically effective dose of the inhibitor ofthis invention is contemplated to be in the range of about 0.1 μg toabout 100 mg per kg body weight per application. It is contemplated thatthe duration of each application of the inhibitor will be in the rangeof 12 to 24 hours of continuous administration. The characteristics ofthe carrier or other material will depend on the route ofadministration.

[0069] The amount of inhibitor in the pharmaceutical composition of thepresent invention will depend upon the nature and severity of thecondition being treated, and on the nature of prior treatments which thepatient has undergone. Ultimately, the attending physician will decidethe amount of inhibitor with which to treat each individual patient.Initially, the attending physician will administer low doses ofinhibitor and observe the patient's response. Larger doses of inhibitormay be administered until the optimal therapeutic effect is obtained forthe patient, and at that point the dosage is not increased further.

[0070] Administration is preferably intravenous, but other known methodsof administration for anti-inflammatory agents may be used.Administration of the anti-inflammatory compounds identified by themethod of the invention can be carried out in a variety of conventionalways. For example, for topical administration, the anti-inflammatorycompound of the invention will be in the form of a pyrogen-free,dermatologically acceptable liquid or semi-solid formulation such as anointment, cream, lotion, foam or gel. The preparation of such topicallyapplied formulations is within the skill in the art. Gel formulationshould contain, in addition to the anti-inflammatory compound, about 2to about 5,5% W/W of a gelling agent. The gelling agent may alsofunction to stabilize the active ingredient and preferably should bewater soluble. The formulation should also contain about 2% W/V of abactericidal agent and a buffering agent. Exemplary gels include ethyl,methyl, and propyl celluloses. Preferred gels includecarboxypolymethylene such as Carbopol (934P; B. F. Goodrich),hydroxypropyl methylcellulose phthalates such as Methocel (K100Mpremium; Merril Dow), cellulose gums such as Blanose (7HF; Aqualon,U.K.), xanthan gums such as Keltrol (TF; Kelko International),hydroxyethyl cellulose oxides such as Polyox (WSR 303; Union Carbide),propylene glycols, polyethylene glycols and mixtures thereof. IfCarbopol is used, a neutralizing agent, such as NaOH, is also requiredin order to maintain pH in the desired range of about 7 to about 8 andmost desirably at about 7.5. Exemplary preferred bactericidal agentsinclude steryl alcohols, especially benzyl alcohol. The buffering agentcan be any of those already known in the art as useful in preparingmedicinal formulations, for example 20 mM phosphate buffer, pH 7.5.

[0071] Cutaneous or subcutaneous injection may also be employed and inthat case the anti-inflammatory compound of the invention will be in theform of pyrogen-free, parenterally acceptable aqueous solutions. Thepreparation of such parenterally acceptable solutions, having due regardto pH, isotonicity, stability, and the like, is within the skill in theart.

[0072] Intravenous injection may be employed, wherein theanti-inflammatory compound of the invention will be in the form ofpyrogen-free, parenterally acceptable aqueous solutions. A preferredpharmaceutical composition for intravenous injection should contain, inaddition to the anti-inflammatory compound, an isotonic vehicle such asSodium Chloride Injection, Ringer's Injection, Dextrose Injection,Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, orother vehicle as known in the art. The pharmaceutical compositionaccording to the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additive known to thoseof skill in the art.

[0073] The amount of anti-inflammatory compound in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of anti-inflammatory compound withwhich to treat each individual patient.

[0074] Anti-inflammatory compounds identified using the method of thepresent invention may be administered alone or in combination with otheranti-inflammation agents and therapies.

EXAMPLE 1 Purification of Calcium Independent cPLA₂

[0075] A) Preparation of CHO-Dukx Cytosolic Fraction

[0076] CHO cells, approximately 5×10¹¹ cells from a 250 L culture, wereconcentrated by centrifugation and rinsed once with phosphate-bufferedsaline and reconcentrated. the cell slurry was frozen in liquid nitrogenand stored at −80° C. at 4×10¹¹ cells/kg of pellet. The CHO pellets wereprocessed in 0.5 kg batches by thawing the cells in 1.2 L of 20 mMimidazol pH 7.5, 0.25M sucrose, 2 mM EDTA, 2 mM EGTA, 1 μg/ml leupeptin,5 μg/ml aprotinin, 5 mM DTT and 1 mM PMSF (“Extraction Buffer”). Thecells were transferred to a Parr bomb at 4° C. and pressurized at 600psi for 5 minutes and lysed by releasing the pressure. The supernatantwas centrifuged at 10,000×g for 30 minutes and subsequently at 100,000×gfor 60 minutes.

[0077] B) DEAE Anion Exchange Chromatography

[0078] The cytosolic fraction (10 gm protein) was diluted to 5 mg/mlwith 20 mM imidazol pH 7.5, 5 mM DTT, 1 mM EDTA and 1 mM EGTA (Buffer A)and applied to a 1 L column of DEAE toyopearl equilibrated in buffer Aat 16 ml/min. The column was washed to background absorbance (A₂₈₀) withbuffer A and developed with a gradient of 0-0.5M NaCl in buffer A over240 minutes with one minute fractions. The first activity peak at100-150 mM NaCl was collected.

[0079] C) Hydrophobic Interaction and Heparin Toyopearl Chromatography

[0080] The DEAE fractions (4 gm of protein at 3 mg/ml) were made 0.5M inammonium sulfate and applied at 10 ml/min to a 300 ml phenyl toyopearlcolumn equilibrated in buffer A containing 0.5M ammonium sulfate. Thecolumn was washed to background absorbance (A₂₈₀). The column was thendeveloped with a gradient of 0.5-0.2M (15 minutes) then 0.2-0.0 Mammonium sulfate (85 minutes). The column was then connected in tandemto a 10 ml heparin column equilibrated in buffer A and elution wascontinued for 18 hours at 1.5 ml/min with buffer A. The phenyl columnwas disconnected and the activity was eluted from the heparin column byapplying 0.5M NaCl in buffer A at 2 ml/min.

[0081] D) Chromatofocussing Chromatography

[0082] A portion of the above active fractions (16 mg) was dialyzedexhaustively against 20 mM Bis-Tris pH 7, 10% glycerol, 1M urea and 5 mMDTT and applied at 0.5 ml/min to a Mono P 5/20 column equilibrated withthe same buffer. The column was washed with the same buffer tobackground absorbance (A₂₈₀) and a pH gradient was established byapplying 10% polybuffer 74 pH 5, 10% glycerol, 1M urea and 5 mM DTT.

[0083] The relative purification of the enzyme of the present inventionat each step of the foregoing purification scheme is summarized in TableII. TABLE II Fold Protein Activity Specific Activity Purifi- Yield Step(mg) (u**) (u/mg) cation (%) cytosolic 126,000 2050 0.016 — — extract*DEAE  16,000 1264 0.079    5 60 phenyl/    193  90 0.46   30 4.5 heparinMono P 0.1-0.2  14 140 8,000 0.7

[0084] The phospholipase can be further purified by the following steps:

[0085] E) Heparin Chromatography

[0086] The sample from (D) above is applied at 0.5 ml/min onto a heparincolumn (maximum capacity lOmg protein/ml of resin) equilibrated inbuffer A. The activity is eluted by 0.4M NaCl in buffer A.

[0087] F) Size Exclusion Chromatography

[0088] The active fractions from the heparin column are applied to twoTSK G3000SW_(XL) columns (7.8 mm×30 cm) linked in tandem equilibratedwith 150 mM NaCl in buffer A at 0.3 ml/min. Phospholipase activityelutes in the 250-350 kD size range.

[0089] Recombinant enzyme may also be purified in accordance with thisexample.

EXAMPLE 2 Amino Acid Sequencing

[0090] A portion (63 μg total protein) of the Mono P active fractionswas concentrated on a heparin column, as described above. The sample,0.36 ml was mixed with an equal volume of buffer A and 10% SDS, 10 μland concentrated to 40 μl on an Amicon-30 microconcentrator. The samplewas diluted with buffer A, 100 μl, concentrated to 60 μl and dilutedwith Laemmli buffer (2×), 40 μl. The solution was boiled for 5 minutesand loaded in three aliquots on a 4-20% gradient SDS-PAGE mini gel. Thesample was electophoresed for two hours at 120 v, stained for 20 minutesin 0.2% Blue R-250, 20% methanol and 0.5% acetic acid and destained in30% methanol (Rosenfeld et. al. Anal. Biochem. 203, pp. 173-179, 1992).Briefly, the protein bands corresponding to the phospholipase wereexcised from the gel with a razor blade and washed with 4 150 μlaliquots of 200 mM NH₄HCO₃, 50% acetonitrile, for a total of 2 hours.The gel pieces were allowed to air dry for approximately 5 minutes, thenpartially rehydrated with 1 μl of 200 mM NH₄HCO₃, 0.02% Tween 20(Pierce) and 2 μl of 0.25 μg/μl trypsin (Promega). Gel slices wereplaced into the bottom of 500 μl mini-Eppendorf tubes, covered with 30μl 200 mM NH₄HCO₃, and incubated at 37 C. for 15 hours. After 1-2minutes of centrifugation in an Eppendorf microfuge, the supernatantswere removed and saved. Peptides in the gel slices were extracted byagitation for a total of 40 minutes with 2 100 μl aliquots of 60%acetonitrile, 0.1% TFA. The extracts were combined with the previoussupernatant. The volume was reduced by lyophilization to about 150 μl,and then the sample was diluted with 750 μl 0.1% TFA. Peptide maps wererun on an ABI 130A Separation System HPLC and an ABI 30×2.1 mm RP-300column. The gradient used was as follows: 0-13.5 minutes 0% B, 13.5-63.5minutes 0-100% B and 63.5-68.5 minutes 100% B, where A is 0.1% TFA and Bis 0.085% TFA, 70% acetonitrile. Peptides were then sequenced on an ABI470A gas-phase sequencer.

EXAMPLE 3 Phopholipase Assays

[0091] 1. sn-2 Hydrolysis Assays

[0092] A) Liposome: The lipid, e.g.1-palmitoyl-2-[¹⁴C]arachidonyl-sn-glycero-3-phosphocholine (PAPC), 55mCi/mmol, was dried under a stream of nitrogen and solubilized inethanol. The assay buffer contained 100 mM Tris-HCl pH 7, 4 mM EDTA, 4mM EGTA, 10% glycerol and 25 μM of labelled PAPC, where the volume ofethanol added was no more than 10% of the final assay volume. Thereaction was incubated for 30 minutes at 37° C. and quenched by theaddition of two volumes of heptane:isopropanol:0.5M sulfuric acid(105:20:1 v/v). Half of the organic was applied to a disposable silicagel column in a vacuum manifold positioned over a scintillation vial,and the free arachidonic was eluted by the addition of ethyl ether (1ml). The level of radioactivity was measured by liquid scintillation.

[0093] Variations on this assay replace EDTA and EGTA with 10 mM CaCl₂.

[0094] B) Mixed Micelle Basic: The lipid was dried down as in (A) and tothis was added the assay buffer consisting of 80 mM glycine pH 9, 5 mMCaCl₂ or 5 mM EDTA, 10% or 70% glycerol and 200 μl triton X-100. Themixture was then sonicated for 30-60 seconds at 4° C. to form mixedmicelles.

[0095] C) Mixed Micelle Neutral: As for (B) except 100 mM Tris-HCl pH 7was used instead of glycine as the buffer.

[0096] 2. sn-1 Hydrolysis Assays

[0097] Sn-1 hydrolysis assays are performed as described above for sn-1hydrolysis, but using phospholipids labelled at the sn-1 substituent,e.g. 1-[¹⁴C]-palmitoyl-2-arachidonyl-sn-glycero-3-phophocholine.

EXAMPLE 4 Cloning of Calcium Independent cPLA₂/B

[0098] A) cDNA Library Construction

[0099] Total RNA was first prepared from 2×10⁸ CHO-DUX cells using theRNAgents total RNA kit (Promega, Madison, Wis.) and further purifiedusing the PolyATract mRNA Isolation System (Promega) to yield 13.2 μgpolyA+ mRNA. Double stranded cDNA was prepared by the Superscript ChoiceSystem (Gibco/BRL, Gaithersburg, Md.) starting with 2 μg of CHO-DUX mRNAand using oligo dT primer. The cDNA was modified at both ends byaddition of an EcoRI adapter/linker provided by the kit. These fragmentswere then ligated into the predigested lambda ZAPII/EcoRI vector, andpackaged into phage particles with Gigapack Gold packaging extracts(Stratagene, La Jolla, Calif.).

[0100] B) Oligonucleotide Probe Design

[0101] Several of the peptide sequences determined for the purifiedcalcium independent PLA₂/B were selected to design oligonucleotideprobes. The amino acid sequence from amino acid 361 to 367 of SEQ IDNO:2 was used to design two degenerate oligonucleotide pools of 17residues each. Pool 1 is 8-fold degenerate representing the sense strandfor amino acids 361 to 366 of SEQ ID NO:2, and pool 2 is 12-folddegenerate representing the antisense strand for amino acids 362-367 ofSEQ ID NO:2. Two other degenerate pools were also made from othersequences. Pool 3 is 32-fold degenerate and represents the sense strandfor amino acids 490 to 495 of SEQ ID NO:2, and pool 4 is 64-folddegenerate representing the antisense strand for amino acids 513 to 518of SEQ ID NO:2.

[0102] C) Library Screening

[0103] Approximately 400,000 recombinant bacteriophage from the CHO-DUXcDNA library were plated and duplicate nitrocellulose filters wereprepared. One set of filters was hybridized with pool 1 and the otherwith pool 2 using tetramethylammonium chloride buffer conditions (Jacobset al., Nature, 1985, 313, 806). Twelve positive bacteriophages wereidentified and plated for further analysis. Three sets of nitrocellulosefilters were prepared from this plating and hybridized with pools 2, 3and 4, to represent the three peptide sequences from which probes weredesigned. Several clones were positive for all three pools. Individualbacteriophage plaques were eluted and ampicillin resistant plasmidcolonies were prepared following the manufacturer's protocols(Stratagene). Plasmid DNA was prepared for clones 9, 17, 31 and 49, andrestriction digests revealed 3.0 kb inserts. Analysis of a portion ofthe DNA sequence in these clones confirmed that they contained severalcPLA₂/B peptide sequences and represented the complete coding region ofthe gene. Clone 9 was selected for complete DNA sequence determination.The sequence of clone 9 is reported as SEQ ID NO:1.

[0104] Clone 9 was deposited with ATCC on Jul. 27, 1994 as accessionnumber 69669.

EXAMPLE 5

[0105] Expression of Recombinant cPLA₂/B

[0106] A) Expression in COS Cells

[0107] Clone 9 from Example 4 was excised inserted into a SalI site thatwas engineered into the EcoRI site of the COS expression vector, PMT-2,a beta lactamase derivative of p91023 (Wong et al., Science, 1985, 228,810). 8 μg of plasmid DNA was then transfected into 1×10⁶ COS cells in a10 cm dish by the DEAE dextran protocol (Sompayrac et al., Proc. Natl.Acad. Sci. USA, 1981, 78, 7575) with the addition of a 0.1 mMchloroquine to the transfection medium, followed by incubation for 3hours at 37° C. The cells were grown in conventional media (DME, 10%fetal calf serum). At 40-48 hours post-transfection the cells werewashed twice and then incubated at 37° C. in PBS, 1 mM EDTA (5 ml). Thecells were then collected by centrifugation, resuspended in ExtractionBuffer (0.5 ml), and lysed by 20 strokes in a Dounce at 4° C. The lysatewas clarified by centrifugation and 10-50 μl of the cytosolic fractionwas assayed in the neutral and pH 9 mixed micelle assays.

[0108] In a further experiment, COS cells were transiently transfectedaccording to established procedures (Kaufman et al.). After 40-48 hourspost-tranmsfection the cells wer labelled with ³⁵S-methionine, 200 μCiper 10 cm plate, for one hour and the cells were lysed in NP-40 lysisbuffer (Kaufman et al.). The cell lysates were analyzed by SDS-PAGE on a4-20% reducing gel where equal counts were loaded per lane. There was anadditional protein band at 84-86 kD in the lysates from cellstransfected with clones 9, 31 and 49, but not in controls (see FIG. 7).

[0109] B) Expression in CHO Cells

[0110] A single plasmid bearing both the cPLA₂/B encoding sequence and aDHFR gene, or two separate plasmids bearing such sequences, areintroduced into DHFR-deficient CHO cells (such as Dukx-BII) by calciumphosphate coprecipitation and transfection. DHFR expressingtransformants are selected for growth in alpha media with dialyzed fetalcalf serum. Transformants are checked for expression of recombinantenzyme by bioassay, immunoassay or RNA blotting and positive pools aresubsequently selected for amplification by growth in increasingconcentrations of methotrexate (MTX) (sequential steps in 0.02, 0.2, 1.0and 5 μM MTX) as described in Kaufman et al., Mol. Cell Biol., 1983, 5,1750. The amplified lines are cloned and recombinant enzyme expressionis monitored by the mixed micelle assay. Recombinant enzyme expressionis expected to increase with increasing levels of MTX resistance.

EXAMPLE 6

[0111] Mutagenesis of Serine Residues

[0112] Ser252 and Ser465 of the murine cPLA₂/B amino acid sequence weremutated to alanine residues using the Chamelon Mutagenesis kit(Stratagene) using oligonucleotides CATGGGACCCGCTGGCTTTCC (SEQ ID NO:24)and GGCAGGAACCGCCACTGGGGGC (SEQ ID NO:25), respectively. PLA₂ activitywas abrogated by changing Ser465 to Ala in the lipase consensus sequence(GXSXGG) surrounding that residue. Although Ser252 is found in a partiallipase motif, mutagenesis did not result in loss of activity. Moreover,Ser465, and the lipase consensus sequence surrounding this residue, areconserved in the human sequence (see amino acids 462-467 of SEQ ID NO:21and 463-468 of SEQ ID NO:23), while Ser252 is not. On this basis, it isbelieved that this conserved serine residue is required for activity.

[0113] Patent and literature references cited herein are incorporated byreference as if fully set forth.

1 25 2935 base pairs nucleic acid double linear cDNA NO NO CDS 96..23521 GCGGCCGCGT CGACGAAGTA AGCGGGCGGA GAAGTGCTGA GTAAGCCGAG AGTAAGGGGG 60CAGGCTGTCC CCCCCCCCCA CCTGCCCCAC GGAGG ATG CAG TTC TTC GGA CGC 113 MetGln Phe Phe Gly Arg 1 5 CTT GTC AAC ACC CTC AGT AGT GTC ACC AAC TTG TTCTCA AAC CCA TTC 161 Leu Val Asn Thr Leu Ser Ser Val Thr Asn Leu Phe SerAsn Pro Phe 10 15 20 CGG GTG AAG GAG ATA TCT GTG GCT GAC TAT ACC TCA CATGAA CGT GTT 209 Arg Val Lys Glu Ile Ser Val Ala Asp Tyr Thr Ser His GluArg Val 25 30 35 CGA GAG GAA GGG CAG CTG ATC CTG TTC CAG AAT GCT TCC AATCGC ACC 257 Arg Glu Glu Gly Gln Leu Ile Leu Phe Gln Asn Ala Ser Asn ArgThr 40 45 50 TGG GAC TGC ATC CTG GTC AGC CCT AGG AAC CCA CAT AGT GGC TTCCGA 305 Trp Asp Cys Ile Leu Val Ser Pro Arg Asn Pro His Ser Gly Phe Arg55 60 65 70 CTC TTC CAG CTG GAG TCA GAG GCA GAT GCC CTG GTG AAC TTC CAGCAG 353 Leu Phe Gln Leu Glu Ser Glu Ala Asp Ala Leu Val Asn Phe Gln Gln75 80 85 TTC TCC TCC CAG CTG CCA CCC TTC TAC GAG AGC TCT GTG CAG GTC CTG401 Phe Ser Ser Gln Leu Pro Pro Phe Tyr Glu Ser Ser Val Gln Val Leu 9095 100 CAT GTG GAG GTG CTG CAG CAC CTG TCT GAC CTG ATC CGA AGC CAC CCC449 His Val Glu Val Leu Gln His Leu Ser Asp Leu Ile Arg Ser His Pro 105110 115 AGC TGG ACG GTG ACA CAC CTG GCG GTG GAG CTT GGC ATT CGG GAG TGC497 Ser Trp Thr Val Thr His Leu Ala Val Glu Leu Gly Ile Arg Glu Cys 120125 130 TTC CAC CAC AGC CGC ATC ATC AGC TGC GCC AAC AGC ACA GAG AAT GAG545 Phe His His Ser Arg Ile Ile Ser Cys Ala Asn Ser Thr Glu Asn Glu 135140 145 150 GAG GGC TGC ACC CCA CTG CAT TTG GCA TGC CGC AAG GGT GAC AGTGAG 593 Glu Gly Cys Thr Pro Leu His Leu Ala Cys Arg Lys Gly Asp Ser Glu155 160 165 ATC CTG GTG GAG TTG GTA CAG TAC TGC CAT GCC CAA ATG GAT GTCACT 641 Ile Leu Val Glu Leu Val Gln Tyr Cys His Ala Gln Met Asp Val Thr170 175 180 GAC AAC AAA GGA GAG ACG GCC TTC CAT TAC GCT GTA CAA GGG GACAAT 689 Asp Asn Lys Gly Glu Thr Ala Phe His Tyr Ala Val Gln Gly Asp Asn185 190 195 TCC CAG GTG CTG CAG CTC CTA GGA AAG AAC GCC TCA GCT GGC CTGAAC 737 Ser Gln Val Leu Gln Leu Leu Gly Lys Asn Ala Ser Ala Gly Leu Asn200 205 210 CAG GTG AAC AAA CAA GGG CTA ACT CCA CTG CAC CTG GCC TGC CAGATG 785 Gln Val Asn Lys Gln Gly Leu Thr Pro Leu His Leu Ala Cys Gln Met215 220 225 230 GGG AAG CAG GAG ATG GTA CGC GTC CTG CTG CTT TGC AAT GCCCGC TGC 833 Gly Lys Gln Glu Met Val Arg Val Leu Leu Leu Cys Asn Ala ArgCys 235 240 245 AAC GTC ATG GGA CCC AGT GGC TTT CCC ATC CAC ACA GCC ATGAAG TTC 881 Asn Val Met Gly Pro Ser Gly Phe Pro Ile His Thr Ala Met LysPhe 250 255 260 TCC CAG AAG GGG TGT GCT GAA ATG ATT ATC AGC ATG GAC AGCAGC CAG 929 Ser Gln Lys Gly Cys Ala Glu Met Ile Ile Ser Met Asp Ser SerGln 265 270 275 ATC CAC AGC AAG GAT CCT CGC TAT GGA GCC AGC CCG CTC CACTGG GCC 977 Ile His Ser Lys Asp Pro Arg Tyr Gly Ala Ser Pro Leu His TrpAla 280 285 290 AAG AAT GCC GAG ATG GCC CGG ATG CTG CTG AAG CGG GGA TGTGAT GTG 1025 Lys Asn Ala Glu Met Ala Arg Met Leu Leu Lys Arg Gly Cys AspVal 295 300 305 310 GAC AGC ACA AGC GCT GCG GGG AAC ACA GCC CTG CAT GTGGCA GTG ATG 1073 Asp Ser Thr Ser Ala Ala Gly Asn Thr Ala Leu His Val AlaVal Met 315 320 325 CGG AAC CGC TTT GAC TGC GTC ATG GTG CTG CTG ACC TACGGG GCC AAC 1121 Arg Asn Arg Phe Asp Cys Val Met Val Leu Leu Thr Tyr GlyAla Asn 330 335 340 GCA GGC ACC CCA GGG GAG CAT GGG AAC ACG CCG CTG CACCTG GCC ATC 1169 Ala Gly Thr Pro Gly Glu His Gly Asn Thr Pro Leu His LeuAla Ile 345 350 355 TCG AAA GAT AAC ATG GAG ATG ATC AAA GCC CTC ATT GTATTT GGG GCA 1217 Ser Lys Asp Asn Met Glu Met Ile Lys Ala Leu Ile Val PheGly Ala 360 365 370 GAA GTG GAT ACC CCA AAT GAC TTT GGG GAG ACT CCT GCCTTC ATG GCC 1265 Glu Val Asp Thr Pro Asn Asp Phe Gly Glu Thr Pro Ala PheMet Ala 375 380 385 390 TCC AAG ATC AGC AAA CAG CTT CAG GAC CTC ATG CCCATC TCC CGA GCC 1313 Ser Lys Ile Ser Lys Gln Leu Gln Asp Leu Met Pro IleSer Arg Ala 395 400 405 CGG AAG CCA GCA TTC ATC CTG AGC TCC ATG AGG GATGAG AAG CGA ATC 1361 Arg Lys Pro Ala Phe Ile Leu Ser Ser Met Arg Asp GluLys Arg Ile 410 415 420 CAT GAT CAC CTG CTC TGC CTG GAC GGA GGG GGC GTGAAA GGC CTG GTC 1409 His Asp His Leu Leu Cys Leu Asp Gly Gly Gly Val LysGly Leu Val 425 430 435 ATC ATC CAA CTC CTC ATT GCC ATC GAG AAG GCC TCAGGT GTG GCC ACC 1457 Ile Ile Gln Leu Leu Ile Ala Ile Glu Lys Ala Ser GlyVal Ala Thr 440 445 450 AAG GAC CTC TTC GAC TGG GTG GCA GGA ACC AGC ACTGGG GGC ATC CTG 1505 Lys Asp Leu Phe Asp Trp Val Ala Gly Thr Ser Thr GlyGly Ile Leu 455 460 465 470 GCC CTG GCC ATT CTG CAC AGT AAG TCC ATG GCCTAT ATG CGT GGT GTG 1553 Ala Leu Ala Ile Leu His Ser Lys Ser Met Ala TyrMet Arg Gly Val 475 480 485 TAC TTC CGT ATG AAA GAT GAG GTG TTT CGG GGCTCA CGG CCC TAT GAG 1601 Tyr Phe Arg Met Lys Asp Glu Val Phe Arg Gly SerArg Pro Tyr Glu 490 495 500 TCT GGA CCC CTG GAG GAG TTC CTG AAG CGG GAGTTT GGG GAG CAC ACC 1649 Ser Gly Pro Leu Glu Glu Phe Leu Lys Arg Glu PheGly Glu His Thr 505 510 515 AAG ATG ACA GAT GTC AAA AAA CCC AAG GTG ATGCTC ACA GGG ACA CTG 1697 Lys Met Thr Asp Val Lys Lys Pro Lys Val Met LeuThr Gly Thr Leu 520 525 530 TCT GAC CGG CAG CCA GCA GAG CTC CAC CTG TTCCGC AAT TAC GAT GCT 1745 Ser Asp Arg Gln Pro Ala Glu Leu His Leu Phe ArgAsn Tyr Asp Ala 535 540 545 550 CCA GAG GTC ATT CGG GAA CCT CGC TTC AACCAA AAC ATT AAC CTG AAG 1793 Pro Glu Val Ile Arg Glu Pro Arg Phe Asn GlnAsn Ile Asn Leu Lys 555 560 565 CCG CCA ACT CAG CCT GCA GAC CAA CTG GTATGG CGA GCA GCC CGG AGC 1841 Pro Pro Thr Gln Pro Ala Asp Gln Leu Val TrpArg Ala Ala Arg Ser 570 575 580 AGT GGG GCA GCC CCA ACC TAC TTC CGG CCCAAT GGA CGT TTC CTG GAT 1889 Ser Gly Ala Ala Pro Thr Tyr Phe Arg Pro AsnGly Arg Phe Leu Asp 585 590 595 GGT GGG CTG CTG GCC AAC AAC CCC ACA CTAGAT GCC ATG ACT GAA ATC 1937 Gly Gly Leu Leu Ala Asn Asn Pro Thr Leu AspAla Met Thr Glu Ile 600 605 610 CAT GAA TAC AAT CAG GAC ATG ATC CGC AAGGGC CAA GGC AAC AAG GTG 1985 His Glu Tyr Asn Gln Asp Met Ile Arg Lys GlyGln Gly Asn Lys Val 615 620 625 630 AAG AAA CTC TCC ATA GTC GTC TCT CTGGGG ACA GGA AGG TCC CCT CAA 2033 Lys Lys Leu Ser Ile Val Val Ser Leu GlyThr Gly Arg Ser Pro Gln 635 640 645 GTG CCC GTA ACC TGT GTA GAT GTC TTCCGC CCC AGC AAC CCC TGG GAA 2081 Val Pro Val Thr Cys Val Asp Val Phe ArgPro Ser Asn Pro Trp Glu 650 655 660 CTG GCT AAG ACT GTT TTT GGA GCC AAGGAA CTG GGC AAG ATG GTG GTA 2129 Leu Ala Lys Thr Val Phe Gly Ala Lys GluLeu Gly Lys Met Val Val 665 670 675 GAC TGT TGC ACA GAT CCA GAT GGT CGGGCT GTG GAC CGG GCC CGG GCC 2177 Asp Cys Cys Thr Asp Pro Asp Gly Arg AlaVal Asp Arg Ala Arg Ala 680 685 690 TGG AGC GAG ATG GTT GGC ATC CAG TACTTC AGA CTG AAC CCC CAA CTA 2225 Trp Ser Glu Met Val Gly Ile Gln Tyr PheArg Leu Asn Pro Gln Leu 695 700 705 710 GGA TCA GAC ATC ATG CTG GAT GAGGTC AAT GAT GCA GTG CTG GTT AAT 2273 Gly Ser Asp Ile Met Leu Asp Glu ValAsn Asp Ala Val Leu Val Asn 715 720 725 GCC CTC TGG GAG ACA GAA GTC TACATC TAT GAG CAC CGG GAG GAG TTC 2321 Ala Leu Trp Glu Thr Glu Val Tyr IleTyr Glu His Arg Glu Glu Phe 730 735 740 CAG AAG CTT GTC CAA ATG CTG CTGTCG CCC T GAGCTCCAGG CCCTGCTGGC 2372 Gln Lys Leu Val Gln Met Leu Leu SerPro 745 750 AGGGGTGCGC CAGGCTACCC AGCACACTGG GGGCCAAGCT GGGCCAGGCGGCTGTGTCTA 2432 CCTGAGGACT GGGGCTCAGA GCACAAACAG GTTCCCACAA GGCACCTCTCCTGACCCATC 2492 TGCACTTTGC CACTCTAGGC TGAAAGCCCA GAGTTCCCCT CAGCCCCTTTATGTGACTGT 2552 GAAGGACAAC TGGCTCCATC AACTGCCCTA AATATCAGTG AGATCAACACTAAGGTGTCC 2612 AGTGTACCCA GAGGGTTCTT CCAGGGTCCA TGGCCACCAA AGCCCACCCCTTCTTTCCAC 2672 TTCCTGAAGT CAGTGTCTAC AGAAATGGAG TTCCACCCCA TCATCAGGTGAAATCCAGGC 2732 TATTGAAATC CAGTCTGTTC GACTTTGCCC CTCTGCACCT GCCAATCACCCCACCCCTGC 2792 AGCCACCCCA CCTTAAGAGT CCTCCCAGCT CTCAAAGGTC AATCCTGTGCATGTACTCTT 2852 CTCTGGAAGG AGAGTGGGGA GGGGTTCAAG GCCACCTCAA CTGTGAAATAAATGGGTCTA 2912 GACTCAAAAA AAAAAAGTCG ACG 2935 752 amino acids aminoacid linear protein 2 Met Gln Phe Phe Gly Arg Leu Val Asn Thr Leu SerSer Val Thr Asn 1 5 10 15 Leu Phe Ser Asn Pro Phe Arg Val Lys Glu IleSer Val Ala Asp Tyr 20 25 30 Thr Ser His Glu Arg Val Arg Glu Glu Gly GlnLeu Ile Leu Phe Gln 35 40 45 Asn Ala Ser Asn Arg Thr Trp Asp Cys Ile LeuVal Ser Pro Arg Asn 50 55 60 Pro His Ser Gly Phe Arg Leu Phe Gln Leu GluSer Glu Ala Asp Ala 65 70 75 80 Leu Val Asn Phe Gln Gln Phe Ser Ser GlnLeu Pro Pro Phe Tyr Glu 85 90 95 Ser Ser Val Gln Val Leu His Val Glu ValLeu Gln His Leu Ser Asp 100 105 110 Leu Ile Arg Ser His Pro Ser Trp ThrVal Thr His Leu Ala Val Glu 115 120 125 Leu Gly Ile Arg Glu Cys Phe HisHis Ser Arg Ile Ile Ser Cys Ala 130 135 140 Asn Ser Thr Glu Asn Glu GluGly Cys Thr Pro Leu His Leu Ala Cys 145 150 155 160 Arg Lys Gly Asp SerGlu Ile Leu Val Glu Leu Val Gln Tyr Cys His 165 170 175 Ala Gln Met AspVal Thr Asp Asn Lys Gly Glu Thr Ala Phe His Tyr 180 185 190 Ala Val GlnGly Asp Asn Ser Gln Val Leu Gln Leu Leu Gly Lys Asn 195 200 205 Ala SerAla Gly Leu Asn Gln Val Asn Lys Gln Gly Leu Thr Pro Leu 210 215 220 HisLeu Ala Cys Gln Met Gly Lys Gln Glu Met Val Arg Val Leu Leu 225 230 235240 Leu Cys Asn Ala Arg Cys Asn Val Met Gly Pro Ser Gly Phe Pro Ile 245250 255 His Thr Ala Met Lys Phe Ser Gln Lys Gly Cys Ala Glu Met Ile Ile260 265 270 Ser Met Asp Ser Ser Gln Ile His Ser Lys Asp Pro Arg Tyr GlyAla 275 280 285 Ser Pro Leu His Trp Ala Lys Asn Ala Glu Met Ala Arg MetLeu Leu 290 295 300 Lys Arg Gly Cys Asp Val Asp Ser Thr Ser Ala Ala GlyAsn Thr Ala 305 310 315 320 Leu His Val Ala Val Met Arg Asn Arg Phe AspCys Val Met Val Leu 325 330 335 Leu Thr Tyr Gly Ala Asn Ala Gly Thr ProGly Glu His Gly Asn Thr 340 345 350 Pro Leu His Leu Ala Ile Ser Lys AspAsn Met Glu Met Ile Lys Ala 355 360 365 Leu Ile Val Phe Gly Ala Glu ValAsp Thr Pro Asn Asp Phe Gly Glu 370 375 380 Thr Pro Ala Phe Met Ala SerLys Ile Ser Lys Gln Leu Gln Asp Leu 385 390 395 400 Met Pro Ile Ser ArgAla Arg Lys Pro Ala Phe Ile Leu Ser Ser Met 405 410 415 Arg Asp Glu LysArg Ile His Asp His Leu Leu Cys Leu Asp Gly Gly 420 425 430 Gly Val LysGly Leu Val Ile Ile Gln Leu Leu Ile Ala Ile Glu Lys 435 440 445 Ala SerGly Val Ala Thr Lys Asp Leu Phe Asp Trp Val Ala Gly Thr 450 455 460 SerThr Gly Gly Ile Leu Ala Leu Ala Ile Leu His Ser Lys Ser Met 465 470 475480 Ala Tyr Met Arg Gly Val Tyr Phe Arg Met Lys Asp Glu Val Phe Arg 485490 495 Gly Ser Arg Pro Tyr Glu Ser Gly Pro Leu Glu Glu Phe Leu Lys Arg500 505 510 Glu Phe Gly Glu His Thr Lys Met Thr Asp Val Lys Lys Pro LysVal 515 520 525 Met Leu Thr Gly Thr Leu Ser Asp Arg Gln Pro Ala Glu LeuHis Leu 530 535 540 Phe Arg Asn Tyr Asp Ala Pro Glu Val Ile Arg Glu ProArg Phe Asn 545 550 555 560 Gln Asn Ile Asn Leu Lys Pro Pro Thr Gln ProAla Asp Gln Leu Val 565 570 575 Trp Arg Ala Ala Arg Ser Ser Gly Ala AlaPro Thr Tyr Phe Arg Pro 580 585 590 Asn Gly Arg Phe Leu Asp Gly Gly LeuLeu Ala Asn Asn Pro Thr Leu 595 600 605 Asp Ala Met Thr Glu Ile His GluTyr Asn Gln Asp Met Ile Arg Lys 610 615 620 Gly Gln Gly Asn Lys Val LysLys Leu Ser Ile Val Val Ser Leu Gly 625 630 635 640 Thr Gly Arg Ser ProGln Val Pro Val Thr Cys Val Asp Val Phe Arg 645 650 655 Pro Ser Asn ProTrp Glu Leu Ala Lys Thr Val Phe Gly Ala Lys Glu 660 665 670 Leu Gly LysMet Val Val Asp Cys Cys Thr Asp Pro Asp Gly Arg Ala 675 680 685 Val AspArg Ala Arg Ala Trp Ser Glu Met Val Gly Ile Gln Tyr Phe 690 695 700 ArgLeu Asn Pro Gln Leu Gly Ser Asp Ile Met Leu Asp Glu Val Asn 705 710 715720 Asp Ala Val Leu Val Asn Ala Leu Trp Glu Thr Glu Val Tyr Ile Tyr 725730 735 Glu His Arg Glu Glu Phe Gln Lys Leu Val Gln Met Leu Leu Ser Pro740 745 750 7 amino acids amino acid single linear peptide NO 3 Asn ProHis Ser Gly Phe Arg 1 5 11 amino acids amino acid single linear peptideNO 4 Xaa Ala Ser Xaa Gly Leu Asn Gln Val Asn Lys 1 5 10 10 amino acidsamino acid single linear peptide NO 5 Tyr Gly Ala Ser Pro Leu His XaaAla Lys 1 5 10 7 amino acids amino acid single linear peptide NO 6 AspAsn Met Glu Met Ile Lys 1 5 5 amino acids amino acid single linearpeptide NO 7 Gly Val Tyr Phe Arg 1 5 7 amino acids amino acid singlelinear peptide NO 8 Met Lys Asp Glu Val Phe Arg 1 5 7 amino acids aminoacid single linear peptide NO 9 Glu Phe Gly Glu His Thr Lys 1 5 10 aminoacids amino acid single linear peptide NO 10 Val Met Leu Thr Gly Thr LeuSer Asp Arg 1 5 10 9 amino acids amino acid single linear peptide NO 11Xaa Tyr Asp Ala Pro Glu Val Ile Arg 1 5 14 amino acids amino acid singlelinear peptide NO 12 Phe Asn Gln Asn Ile Asn Leu Lys Pro Pro Thr Gln ProAla 1 5 10 11 amino acids amino acid single linear peptide NO 13 Xaa XaaGly Ala Ala Pro Thr Tyr Phe Arg Pro 1 5 10 6 amino acids amino acidsingle linear peptide NO 14 Thr Val Phe Gly Ala Lys 1 5 12 amino acidsamino acid single linear peptide NO 15 Xaa Trp Ser Glu Met Val Gly IleGln Tyr Phe Arg 1 5 10 2012 base pairs nucleic acid double linear cDNANO CDS 43..1224 16 GAATTCCGGG ACGGTGGGGC CTCCCCACCT GCCCCGCAGA AG ATGCAG TTC TTT 54 Met Gln Phe Phe 1 GGC CGC CTG GTC AAT ACC TTC AGT GGC GTCACC AAC TTG TTC TCT AAC 102 Gly Arg Leu Val Asn Thr Phe Ser Gly Val ThrAsn Leu Phe Ser Asn 5 10 15 20 CCA TTC CGG GTG AAG GAG GTG GCT GTG GCCGAC TAC ACC TCG AGT GAC 150 Pro Phe Arg Val Lys Glu Val Ala Val Ala AspTyr Thr Ser Ser Asp 25 30 35 CGA GTT CGG GAG GAA GGG CAG CTG ATT CTG TTCCAG AAC ACT CCC AAC 198 Arg Val Arg Glu Glu Gly Gln Leu Ile Leu Phe GlnAsn Thr Pro Asn 40 45 50 CGC ACC TGG GAC TGC GTC CTG GTC AAC CCC AGG AACTCA CAG AGT GGA 246 Arg Thr Trp Asp Cys Val Leu Val Asn Pro Arg Asn SerGln Ser Gly 55 60 65 TTC CGA CTC TTC CAG CTG GAG TTG GAG GCT GAC GCC CTAGTG AAT TTC 294 Phe Arg Leu Phe Gln Leu Glu Leu Glu Ala Asp Ala Leu ValAsn Phe 70 75 80 CAT CAG TAT TCT TCC CAG CTG CTA CCC TTC TAT GAG AGC TCCCCT CAG 342 His Gln Tyr Ser Ser Gln Leu Leu Pro Phe Tyr Glu Ser Ser ProGln 85 90 95 100 GTC CTG CAC ACT GAG GTC CTG CAG CAC CTG ACC GAC CTC ATCCGT AAC 390 Val Leu His Thr Glu Val Leu Gln His Leu Thr Asp Leu Ile ArgAsn 105 110 115 CAC CCC AGC TGG TCA GTG GCC CAC CTG GCT GTG GAG CTA GGGATC CGC 438 His Pro Ser Trp Ser Val Ala His Leu Ala Val Glu Leu Gly IleArg 120 125 130 GAG TGC TTC CAT CAC AGC CGT ATC ATC AGC TGT GCC AAT TGCGCG GAG 486 Glu Cys Phe His His Ser Arg Ile Ile Ser Cys Ala Asn Cys AlaGlu 135 140 145 AAC GAG GAG GGC TGC ACA CCC CTG CAC CTG GCC TGC CGC AAGGGT GAT 534 Asn Glu Glu Gly Cys Thr Pro Leu His Leu Ala Cys Arg Lys GlyAsp 150 155 160 GGG GAG ATC CTG GTG GAG CTG GTG CAG TAC TGC CAC ACT CAGATG GAT 582 Gly Glu Ile Leu Val Glu Leu Val Gln Tyr Cys His Thr Gln MetAsp 165 170 175 180 GTC ACC GAC TAC AAG GGA GAG ACC GTC TTC CAT TAT GCTGTC CAG GGT 630 Val Thr Asp Tyr Lys Gly Glu Thr Val Phe His Tyr Ala ValGln Gly 185 190 195 GAC AAT TCT CAG GTG CTG CAG CTC CTT GGA AGG AAC GCAGTG GCT GGC 678 Asp Asn Ser Gln Val Leu Gln Leu Leu Gly Arg Asn Ala ValAla Gly 200 205 210 CTG AAC CAG GTG AAT AAC CAA GGG CTG ACC CCG CTG CACCTG GCC TGC 726 Leu Asn Gln Val Asn Asn Gln Gly Leu Thr Pro Leu His LeuAla Cys 215 220 225 CAG CTG GGG AAG CAG GAG ATG GTC CGC GTG CTG CTG CTGTGC AAT GCT 774 Gln Leu Gly Lys Gln Glu Met Val Arg Val Leu Leu Leu CysAsn Ala 230 235 240 CGG TGC AAC ATC ATG GGC CCC AAC GGC TAC CCC ATC CACTCG GCC ATG 822 Arg Cys Asn Ile Met Gly Pro Asn Gly Tyr Pro Ile His SerAla Met 245 250 255 260 AAG TTC TCT CAG AAG GGG TGT GCG GAG ATG ATC ATCAGC ATG GAC AGC 870 Lys Phe Ser Gln Lys Gly Cys Ala Glu Met Ile Ile SerMet Asp Ser 265 270 275 AGC CAG ATC CAC AGC AAA GAC CCC CGT TAC GGA GCCAGC CCC CTC CAC 918 Ser Gln Ile His Ser Lys Asp Pro Arg Tyr Gly Ala SerPro Leu His 280 285 290 TGG GCC AAG AAC GCA GAG ATG GCC CGC ATG CTG CTGAAA CGG GGC TGC 966 Trp Ala Lys Asn Ala Glu Met Ala Arg Met Leu Leu LysArg Gly Cys 295 300 305 AAC GTG AAC AGC ACC AGC TCC GCG GGG AAC ACG GCCCTG CAC GTG GGG 1014 Asn Val Asn Ser Thr Ser Ser Ala Gly Asn Thr Ala LeuHis Val Gly 310 315 320 GTG ATG CGC AAC CGC TTC GAC TGT GCC ATA GTG CTGCTG ACC CAC GGG 1062 Val Met Arg Asn Arg Phe Asp Cys Ala Ile Val Leu LeuThr His Gly 325 330 335 340 GCC AAC GCG GAT GCC CGC GGA GAG CAC GGC AACACC CCG CTG CAC CTG 1110 Ala Asn Ala Asp Ala Arg Gly Glu His Gly Asn ThrPro Leu His Leu 345 350 355 GCC ATG TCG AAA GAC AAC GTG GAG ATG ATC AAGGCC CTC ATC GTG TTC 1158 Ala Met Ser Lys Asp Asn Val Glu Met Ile Lys AlaLeu Ile Val Phe 360 365 370 GGA GCA GAA GTG GAC ACC CCG AAT GAC TTT GGGGAG ACT CCT ACA TTC 1206 Gly Ala Glu Val Asp Thr Pro Asn Asp Phe Gly GluThr Pro Thr Phe 375 380 385 CTA GCC TCC AAA ATC GGC AGACTTGTCACCAGGAAGGC GATCTTGACT 1254 Leu Ala Ser Lys Ile Gly 390 CTGCTGAGAACCGTGGGGGC CGAATACTGC TTCCCACCCA TCCACGGGGT CCCCGCGGAG 1314 CAGGGCTCTGCAGCGCCACA TCATCCCTTC TCCCTGGAAA GAGCTCAGCC CCCACCGATC 1374 AGCCTAAACAACCTAGGCAG TCACCCAAGC CAGGCCGGAT GGTGGGCCTG GGGTGCGGCG 1434 TCAGATGGGTAACGCCCTGG GCCTGGAGAG GCCACCGAGC CTAGCCATGC GGCATTAGCT 1494 CTAGCTCTCACTCCCTAATC CGTCCTTCTT AGCTGCGCAC ACACCACACG CCCCCTCCCC 1554 TGCACCCTGTCCCCGGCCTC TCTCAGCCAC TCTTCTGCTT CCCTTGTTCA CTGTGCAGCC 1614 GTGTGCCCTGGGGAGGGGGA GACACCGCTT CGCAGCCCTC GGTTCTGCTT TGCTGCTTCT 1674 AGACTCTGCACAGTGGTGGG GGGCTGTCAG AGTTGGGGTC ACGCGGGCTG CTGCACCAGG 1734 CACCTGGGGACTGGGCTGCT TGTCAGGAGG GGCAGCTAGT CAGTTGGGTG GACGTCGGGC 1794 AGGCCTTGGACACAAAGGAA GACATGGACA GAGTGGATGG TGGGCCTGAT CCCGGAGGCC 1854 ACTGGGATTTCCAGACCTGG GATCAGGACG AGGGATGTCT CCTTTCATCC ATGGACTTAA 1914 ACCCCGAGGAACGTCCTGAC TCAGCCTTTT GACTAAATGA CCTTGGGTGA ATTATGGACC 1974 CTCTTAGAGCCTCACCTGTC AATAGGGAAT AAGAATTC 2012 394 amino acids amino acid linearprotein 17 Met Gln Phe Phe Gly Arg Leu Val Asn Thr Phe Ser Gly Val ThrAsn 1 5 10 15 Leu Phe Ser Asn Pro Phe Arg Val Lys Glu Val Ala Val AlaAsp Tyr 20 25 30 Thr Ser Ser Asp Arg Val Arg Glu Glu Gly Gln Leu Ile LeuPhe Gln 35 40 45 Asn Thr Pro Asn Arg Thr Trp Asp Cys Val Leu Val Asn ProArg Asn 50 55 60 Ser Gln Ser Gly Phe Arg Leu Phe Gln Leu Glu Leu Glu AlaAsp Ala 65 70 75 80 Leu Val Asn Phe His Gln Tyr Ser Ser Gln Leu Leu ProPhe Tyr Glu 85 90 95 Ser Ser Pro Gln Val Leu His Thr Glu Val Leu Gln HisLeu Thr Asp 100 105 110 Leu Ile Arg Asn His Pro Ser Trp Ser Val Ala HisLeu Ala Val Glu 115 120 125 Leu Gly Ile Arg Glu Cys Phe His His Ser ArgIle Ile Ser Cys Ala 130 135 140 Asn Cys Ala Glu Asn Glu Glu Gly Cys ThrPro Leu His Leu Ala Cys 145 150 155 160 Arg Lys Gly Asp Gly Glu Ile LeuVal Glu Leu Val Gln Tyr Cys His 165 170 175 Thr Gln Met Asp Val Thr AspTyr Lys Gly Glu Thr Val Phe His Tyr 180 185 190 Ala Val Gln Gly Asp AsnSer Gln Val Leu Gln Leu Leu Gly Arg Asn 195 200 205 Ala Val Ala Gly LeuAsn Gln Val Asn Asn Gln Gly Leu Thr Pro Leu 210 215 220 His Leu Ala CysGln Leu Gly Lys Gln Glu Met Val Arg Val Leu Leu 225 230 235 240 Leu CysAsn Ala Arg Cys Asn Ile Met Gly Pro Asn Gly Tyr Pro Ile 245 250 255 HisSer Ala Met Lys Phe Ser Gln Lys Gly Cys Ala Glu Met Ile Ile 260 265 270Ser Met Asp Ser Ser Gln Ile His Ser Lys Asp Pro Arg Tyr Gly Ala 275 280285 Ser Pro Leu His Trp Ala Lys Asn Ala Glu Met Ala Arg Met Leu Leu 290295 300 Lys Arg Gly Cys Asn Val Asn Ser Thr Ser Ser Ala Gly Asn Thr Ala305 310 315 320 Leu His Val Gly Val Met Arg Asn Arg Phe Asp Cys Ala IleVal Leu 325 330 335 Leu Thr His Gly Ala Asn Ala Asp Ala Arg Gly Glu HisGly Asn Thr 340 345 350 Pro Leu His Leu Ala Met Ser Lys Asp Asn Val GluMet Ile Lys Ala 355 360 365 Leu Ile Val Phe Gly Ala Glu Val Asp Thr ProAsn Asp Phe Gly Glu 370 375 380 Thr Pro Thr Phe Leu Ala Ser Lys Ile Gly385 390 1277 base pairs nucleic acid double linear cDNA NO CDS 396..127118 GAATTCTTAG GCCCCAGGTG GTTATTGCAG CATCGGCTCC GATGCAAGAA GAAGCACTTT 60GTCTGAAGAG GACACGCAAG GGTATTCATG CCTTGGGGTT TCAAGAGGAA GAGATTGAGG 120GGAACCTGGG AGCTGGCTGG GCAGGGTGGG GAGCCCTTCC CAGAGCAGTG GGCCCCCCTT 180TCCACTCCAG CCCATTTCTC TCCTGTGGCC TGTGGCTCAG CTTTCTCCTG GGACAGAGTC 240CTTCCTGTGG GGAAGGGACA GATGACAGGG GGAGTGGGGG GATGAGGGCG TGGCCGTGGG 300CGAGGCACAG CCCAGGTTTG ATCTAGGGAC CTCTGGGGTA GCAGGGCTTG GGGACCCACC 360TGACCACAGC ATGCCCTGCT CTGTGCCTCA CAGAA CTA CAG GAT CTC ATG CAC 413 LeuGln Asp Leu Met His 1 5 ATC TCA CGG GCC CGG AAG CCA GCG TTC ATC CTG GGCTCC ATG AGG GAC 461 Ile Ser Arg Ala Arg Lys Pro Ala Phe Ile Leu Gly SerMet Arg Asp 10 15 20 GAG AAG CGG ACC CAC GAC CAC CTG CTG TGC CTG GAT GGAGGA GGA GTG 509 Glu Lys Arg Thr His Asp His Leu Leu Cys Leu Asp Gly GlyGly Val 25 30 35 AAA GGC CTC ATC ATC ATC CAG CTC CTC ATC GCC ATC GAG AAGGCC TCG 557 Lys Gly Leu Ile Ile Ile Gln Leu Leu Ile Ala Ile Glu Lys AlaSer 40 45 50 GGT GTG GCC ACC AAG GAC CTG TTT GAC TGG GTG GCG GGC ACC AGCACT 605 Gly Val Ala Thr Lys Asp Leu Phe Asp Trp Val Ala Gly Thr Ser Thr55 60 65 70 GGA GGC ATC CTG GCC CTG GCC ATT CTG CAC AGT AAG TCC ATG GCCTAC 653 Gly Gly Ile Leu Ala Leu Ala Ile Leu His Ser Lys Ser Met Ala Tyr75 80 85 ATG CGC GGC ATG TAC TTT CGC ATG AAG GAT GAG GTG TTC CGG GGC TCC701 Met Arg Gly Met Tyr Phe Arg Met Lys Asp Glu Val Phe Arg Gly Ser 9095 100 AGG CCC TAC GAG TCG GGG CCC CTG GAG GAG TTC CTG AAG CGG GAG TTT749 Arg Pro Tyr Glu Ser Gly Pro Leu Glu Glu Phe Leu Lys Arg Glu Phe 105110 115 GGG GAG CAC ACC AAG ATG ACG GAC GTC AGG AAA CCC AAG GTG ATG CTG797 Gly Glu His Thr Lys Met Thr Asp Val Arg Lys Pro Lys Val Met Leu 120125 130 ACA GGG ACA CTG TCT GAC CGG CAG CCG GCT GAA CTC CAC CTC TTC CGG845 Thr Gly Thr Leu Ser Asp Arg Gln Pro Ala Glu Leu His Leu Phe Arg 135140 145 150 AAC TAC GAT GCT CCA GAA ACT GTC CGG GAG CCT CGT TTC AAC CAGAAC 893 Asn Tyr Asp Ala Pro Glu Thr Val Arg Glu Pro Arg Phe Asn Gln Asn155 160 165 GTT AAC CTC AGG CCT CCA GCT CAG CCC TCA GAC CAG CTG GTG TGGCGG 941 Val Asn Leu Arg Pro Pro Ala Gln Pro Ser Asp Gln Leu Val Trp Arg170 175 180 GCG GCC CGA AGC AGC GGG GCA GCT CCT ACT TAC TTC CGA CCC AATGGG 989 Ala Ala Arg Ser Ser Gly Ala Ala Pro Thr Tyr Phe Arg Pro Asn Gly185 190 195 CGC TTC CTG GAC GGT GGG CTG TTG GCC AAC AAC CCC ACG CTG GATGCC 1037 Arg Phe Leu Asp Gly Gly Leu Leu Ala Asn Asn Pro Thr Leu Asp Ala200 205 210 ATG ACC GAG ATC CAT GAG TAC AAT CAG GAC CTG ATC CGC AAG GGTCAG 1085 Met Thr Glu Ile His Glu Tyr Asn Gln Asp Leu Ile Arg Lys Gly Gln215 220 225 230 GCC AAC AAG GTG AAG AAA CTC TCC ATC GTT GTC TCC CTG GGGACA GGG 1133 Ala Asn Lys Val Lys Lys Leu Ser Ile Val Val Ser Leu Gly ThrGly 235 240 245 AGG TCC CCA CAA GTG CCT GTG ACC TGT GTG GAT GTC TTC CGTCCC AGC 1181 Arg Ser Pro Gln Val Pro Val Thr Cys Val Asp Val Phe Arg ProSer 250 255 260 AAC CCC TGG GAG CTG GCC AAG ACT GTT TTT GGG GCC AAG GAACTG GGC 1229 Asn Pro Trp Glu Leu Ala Lys Thr Val Phe Gly Ala Lys Glu LeuGly 265 270 275 AAG ATG GTG GTG GAC TGT TGC ACG GAT CCA GAC GGG CGG CCG1271 Lys Met Val Val Asp Cys Cys Thr Asp Pro Asp Gly Arg Pro 280 285 290GAATTC 1277 292 amino acids amino acid linear protein 19 Leu Gln Asp LeuMet His Ile Ser Arg Ala Arg Lys Pro Ala Phe Ile 1 5 10 15 Leu Gly SerMet Arg Asp Glu Lys Arg Thr His Asp His Leu Leu Cys 20 25 30 Leu Asp GlyGly Gly Val Lys Gly Leu Ile Ile Ile Gln Leu Leu Ile 35 40 45 Ala Ile GluLys Ala Ser Gly Val Ala Thr Lys Asp Leu Phe Asp Trp 50 55 60 Val Ala GlyThr Ser Thr Gly Gly Ile Leu Ala Leu Ala Ile Leu His 65 70 75 80 Ser LysSer Met Ala Tyr Met Arg Gly Met Tyr Phe Arg Met Lys Asp 85 90 95 Glu ValPhe Arg Gly Ser Arg Pro Tyr Glu Ser Gly Pro Leu Glu Glu 100 105 110 PheLeu Lys Arg Glu Phe Gly Glu His Thr Lys Met Thr Asp Val Arg 115 120 125Lys Pro Lys Val Met Leu Thr Gly Thr Leu Ser Asp Arg Gln Pro Ala 130 135140 Glu Leu His Leu Phe Arg Asn Tyr Asp Ala Pro Glu Thr Val Arg Glu 145150 155 160 Pro Arg Phe Asn Gln Asn Val Asn Leu Arg Pro Pro Ala Gln ProSer 165 170 175 Asp Gln Leu Val Trp Arg Ala Ala Arg Ser Ser Gly Ala AlaPro Thr 180 185 190 Tyr Phe Arg Pro Asn Gly Arg Phe Leu Asp Gly Gly LeuLeu Ala Asn 195 200 205 Asn Pro Thr Leu Asp Ala Met Thr Glu Ile His GluTyr Asn Gln Asp 210 215 220 Leu Ile Arg Lys Gly Gln Ala Asn Lys Val LysLys Leu Ser Ile Val 225 230 235 240 Val Ser Leu Gly Thr Gly Arg Ser ProGln Val Pro Val Thr Cys Val 245 250 255 Asp Val Phe Arg Pro Ser Asn ProTrp Glu Leu Ala Lys Thr Val Phe 260 265 270 Gly Ala Lys Glu Leu Gly LysMet Val Val Asp Cys Cys Thr Asp Pro 275 280 285 Asp Gly Arg Pro 290 2109base pairs nucleic acid double linear cDNA NO CDS 43..2103 20 GAATTCCGGGACGGTGGGGC CTCCCCACCT GCCCCGCAGA AG ATG CAG TTC TTT 54 Met Gln Phe Phe 1GGC CGC CTG GTC AAT ACC TTC AGT GGC GTC ACC AAC TTG TTC TCT AAC 102 GlyArg Leu Val Asn Thr Phe Ser Gly Val Thr Asn Leu Phe Ser Asn 5 10 15 20CCA TTC CGG GTG AAG GAG GTG GCT GTG GCC GAC TAC ACC TCG AGT GAC 150 ProPhe Arg Val Lys Glu Val Ala Val Ala Asp Tyr Thr Ser Ser Asp 25 30 35 CGAGTT CGG GAG GAA GGG CAG CTG ATT CTG TTC CAG AAC ACT CCC AAC 198 Arg ValArg Glu Glu Gly Gln Leu Ile Leu Phe Gln Asn Thr Pro Asn 40 45 50 CGC ACCTGG GAC TGC GTC CTG GTC AAC CCC AGG AAC TCA CAG AGT GGA 246 Arg Thr TrpAsp Cys Val Leu Val Asn Pro Arg Asn Ser Gln Ser Gly 55 60 65 TTC CGA CTCTTC CAG CTG GAG TTG GAG GCT GAC GCC CTA GTG AAT TTC 294 Phe Arg Leu PheGln Leu Glu Leu Glu Ala Asp Ala Leu Val Asn Phe 70 75 80 CAT CAG TAT TCTTCC CAG CTG CTA CCC TTC TAT GAG AGC TCC CCT CAG 342 His Gln Tyr Ser SerGln Leu Leu Pro Phe Tyr Glu Ser Ser Pro Gln 85 90 95 100 GTC CTG CAC ACTGAG GTC CTG CAG CAC CTG ACC GAC CTC ATC CGT AAC 390 Val Leu His Thr GluVal Leu Gln His Leu Thr Asp Leu Ile Arg Asn 105 110 115 CAC CCC AGC TGGTCA GTG GCC CAC CTG GCT GTG GAG CTA GGG ATC CGC 438 His Pro Ser Trp SerVal Ala His Leu Ala Val Glu Leu Gly Ile Arg 120 125 130 GAG TGC TTC CATCAC AGC CGT ATC ATC AGC TGT GCC AAT TGC GCG GAG 486 Glu Cys Phe His HisSer Arg Ile Ile Ser Cys Ala Asn Cys Ala Glu 135 140 145 AAC GAG GAG GGCTGC ACA CCC CTG CAC CTG GCC TGC CGC AAG GGT GAT 534 Asn Glu Glu Gly CysThr Pro Leu His Leu Ala Cys Arg Lys Gly Asp 150 155 160 GGG GAG ATC CTGGTG GAG CTG GTG CAG TAC TGC CAC ACT CAG ATG GAT 582 Gly Glu Ile Leu ValGlu Leu Val Gln Tyr Cys His Thr Gln Met Asp 165 170 175 180 GTC ACC GACTAC AAG GGA GAG ACC GTC TTC CAT TAT GCT GTC CAG GGT 630 Val Thr Asp TyrLys Gly Glu Thr Val Phe His Tyr Ala Val Gln Gly 185 190 195 GAC AAT TCTCAG GTG CTG CAG CTC CTT GGA AGG AAC GCA GTG GCT GGC 678 Asp Asn Ser GlnVal Leu Gln Leu Leu Gly Arg Asn Ala Val Ala Gly 200 205 210 CTG AAC CAGGTG AAT AAC CAA GGG CTG ACC CCG CTG CAC CTG GCC TGC 726 Leu Asn Gln ValAsn Asn Gln Gly Leu Thr Pro Leu His Leu Ala Cys 215 220 225 CAG CTG GGGAAG CAG GAG ATG GTC CGC GTG CTG CTG CTG TGC AAT GCT 774 Gln Leu Gly LysGln Glu Met Val Arg Val Leu Leu Leu Cys Asn Ala 230 235 240 CGG TGC AACATC ATG GGC CCC AAC GGC TAC CCC ATC CAC TCG GCC ATG 822 Arg Cys Asn IleMet Gly Pro Asn Gly Tyr Pro Ile His Ser Ala Met 245 250 255 260 AAG TTCTCT CAG AAG GGG TGT GCG GAG ATG ATC ATC AGC ATG GAC AGC 870 Lys Phe SerGln Lys Gly Cys Ala Glu Met Ile Ile Ser Met Asp Ser 265 270 275 AGC CAGATC CAC AGC AAA GAC CCC CGT TAC GGA GCC AGC CCC CTC CAC 918 Ser Gln IleHis Ser Lys Asp Pro Arg Tyr Gly Ala Ser Pro Leu His 280 285 290 TGG GCCAAG AAC GCA GAG ATG GCC CGC ATG CTG CTG AAA CGG GGC TGC 966 Trp Ala LysAsn Ala Glu Met Ala Arg Met Leu Leu Lys Arg Gly Cys 295 300 305 AAC GTGAAC AGC ACC AGC TCC GCG GGG AAC ACG GCC CTG CAC GTG GGG 1014 Asn Val AsnSer Thr Ser Ser Ala Gly Asn Thr Ala Leu His Val Gly 310 315 320 GTG ATGCGC AAC CGC TTC GAC TGT GCC ATA GTG CTG CTG ACC CAC GGG 1062 Val Met ArgAsn Arg Phe Asp Cys Ala Ile Val Leu Leu Thr His Gly 325 330 335 340 GCCAAC GCG GAT GCC CGC GGA GAG CAC GGC AAC ACC CCG CTG CAC CTG 1110 Ala AsnAla Asp Ala Arg Gly Glu His Gly Asn Thr Pro Leu His Leu 345 350 355 GCCATG TCG AAA GAC AAC GTG GAG ATG ATC AAG GCC CTC ATC GTG TTC 1158 Ala MetSer Lys Asp Asn Val Glu Met Ile Lys Ala Leu Ile Val Phe 360 365 370 GGAGCA GAA GTG GAC ACC CCG AAT GAC TTT GGG GAG ACT CCT ACA TTC 1206 Gly AlaGlu Val Asp Thr Pro Asn Asp Phe Gly Glu Thr Pro Thr Phe 375 380 385 CTAGCC TCC AAA ATC GGC AAA CTA CAG GAT CTC ATG CAC ATC TCA CGG 1254 Leu AlaSer Lys Ile Gly Lys Leu Gln Asp Leu Met His Ile Ser Arg 390 395 400 GCCCGG AAG CCA GCG TTC ATC CTG GGC TCC ATG AGG GAC GAG AAG CGG 1302 Ala ArgLys Pro Ala Phe Ile Leu Gly Ser Met Arg Asp Glu Lys Arg 405 410 415 420ACC CAC GAC CAC CTG CTG TGC CTG GAT GGA GGA GGA GTG AAA GGC CTC 1350 ThrHis Asp His Leu Leu Cys Leu Asp Gly Gly Gly Val Lys Gly Leu 425 430 435ATC ATC ATC CAG CTC CTC ATC GCC ATC GAG AAG GCC TCG GGT GTG GCC 1398 IleIle Ile Gln Leu Leu Ile Ala Ile Glu Lys Ala Ser Gly Val Ala 440 445 450ACC AAG GAC CTG TTT GAC TGG GTG GCG GGC ACC AGC ACT GGA GGC ATC 1446 ThrLys Asp Leu Phe Asp Trp Val Ala Gly Thr Ser Thr Gly Gly Ile 455 460 465CTG GCC CTG GCC ATT CTG CAC AGT AAG TCC ATG GCC TAC ATG CGC GGC 1494 LeuAla Leu Ala Ile Leu His Ser Lys Ser Met Ala Tyr Met Arg Gly 470 475 480ATG TAC TTT CGC ATG AAG GAT GAG GTG TTC CGG GGC TCC AGG CCC TAC 1542 MetTyr Phe Arg Met Lys Asp Glu Val Phe Arg Gly Ser Arg Pro Tyr 485 490 495500 GAG TCG GGG CCC CTG GAG GAG TTC CTG AAG CGG GAG TTT GGG GAG CAC 1590Glu Ser Gly Pro Leu Glu Glu Phe Leu Lys Arg Glu Phe Gly Glu His 505 510515 ACC AAG ATG ACG GAC GTC AGG AAA CCC AAG GTG ATG CTG ACA GGG ACA 1638Thr Lys Met Thr Asp Val Arg Lys Pro Lys Val Met Leu Thr Gly Thr 520 525530 CTG TCT GAC CGG CAG CCG GCT GAA CTC CAC CTC TTC CGG AAC TAC GAT 1686Leu Ser Asp Arg Gln Pro Ala Glu Leu His Leu Phe Arg Asn Tyr Asp 535 540545 GCT CCA GAA ACT GTC CGG GAG CCT CGT TTC AAC CAG AAC GTT AAC CTC 1734Ala Pro Glu Thr Val Arg Glu Pro Arg Phe Asn Gln Asn Val Asn Leu 550 555560 AGG CCT CCA GCT CAG CCC TCA GAC CAG CTG GTG TGG CGG GCG GCC CGA 1782Arg Pro Pro Ala Gln Pro Ser Asp Gln Leu Val Trp Arg Ala Ala Arg 565 570575 580 AGC AGC GGG GCA GCT CCT ACT TAC TTC CGA CCC AAT GGG CGC TTC CTG1830 Ser Ser Gly Ala Ala Pro Thr Tyr Phe Arg Pro Asn Gly Arg Phe Leu 585590 595 GAC GGT GGG CTG TTG GCC AAC AAC CCC ACG CTG GAT GCC ATG ACC GAG1878 Asp Gly Gly Leu Leu Ala Asn Asn Pro Thr Leu Asp Ala Met Thr Glu 600605 610 ATC CAT GAG TAC AAT CAG GAC CTG ATC CGC AAG GGT CAG GCC AAC AAG1926 Ile His Glu Tyr Asn Gln Asp Leu Ile Arg Lys Gly Gln Ala Asn Lys 615620 625 GTG AAG AAA CTC TCC ATC GTT GTC TCC CTG GGG ACA GGG AGG TCC CCA1974 Val Lys Lys Leu Ser Ile Val Val Ser Leu Gly Thr Gly Arg Ser Pro 630635 640 CAA GTG CCT GTG ACC TGT GTG GAT GTC TTC CGT CCC AGC AAC CCC TGG2022 Gln Val Pro Val Thr Cys Val Asp Val Phe Arg Pro Ser Asn Pro Trp 645650 655 660 GAG CTG GCC AAG ACT GTT TTT GGG GCC AAG GAA CTG GGC AAG ATGGTG 2070 Glu Leu Ala Lys Thr Val Phe Gly Ala Lys Glu Leu Gly Lys Met Val665 670 675 GTG GAC TGT TGC ACG GAT CCA GAC GGG CGG CCG GAATTC 2109 ValAsp Cys Cys Thr Asp Pro Asp Gly Arg Pro 680 685 687 amino acids aminoacid linear protein 21 Met Gln Phe Phe Gly Arg Leu Val Asn Thr Phe SerGly Val Thr Asn 1 5 10 15 Leu Phe Ser Asn Pro Phe Arg Val Lys Glu ValAla Val Ala Asp Tyr 20 25 30 Thr Ser Ser Asp Arg Val Arg Glu Glu Gly GlnLeu Ile Leu Phe Gln 35 40 45 Asn Thr Pro Asn Arg Thr Trp Asp Cys Val LeuVal Asn Pro Arg Asn 50 55 60 Ser Gln Ser Gly Phe Arg Leu Phe Gln Leu GluLeu Glu Ala Asp Ala 65 70 75 80 Leu Val Asn Phe His Gln Tyr Ser Ser GlnLeu Leu Pro Phe Tyr Glu 85 90 95 Ser Ser Pro Gln Val Leu His Thr Glu ValLeu Gln His Leu Thr Asp 100 105 110 Leu Ile Arg Asn His Pro Ser Trp SerVal Ala His Leu Ala Val Glu 115 120 125 Leu Gly Ile Arg Glu Cys Phe HisHis Ser Arg Ile Ile Ser Cys Ala 130 135 140 Asn Cys Ala Glu Asn Glu GluGly Cys Thr Pro Leu His Leu Ala Cys 145 150 155 160 Arg Lys Gly Asp GlyGlu Ile Leu Val Glu Leu Val Gln Tyr Cys His 165 170 175 Thr Gln Met AspVal Thr Asp Tyr Lys Gly Glu Thr Val Phe His Tyr 180 185 190 Ala Val GlnGly Asp Asn Ser Gln Val Leu Gln Leu Leu Gly Arg Asn 195 200 205 Ala ValAla Gly Leu Asn Gln Val Asn Asn Gln Gly Leu Thr Pro Leu 210 215 220 HisLeu Ala Cys Gln Leu Gly Lys Gln Glu Met Val Arg Val Leu Leu 225 230 235240 Leu Cys Asn Ala Arg Cys Asn Ile Met Gly Pro Asn Gly Tyr Pro Ile 245250 255 His Ser Ala Met Lys Phe Ser Gln Lys Gly Cys Ala Glu Met Ile Ile260 265 270 Ser Met Asp Ser Ser Gln Ile His Ser Lys Asp Pro Arg Tyr GlyAla 275 280 285 Ser Pro Leu His Trp Ala Lys Asn Ala Glu Met Ala Arg MetLeu Leu 290 295 300 Lys Arg Gly Cys Asn Val Asn Ser Thr Ser Ser Ala GlyAsn Thr Ala 305 310 315 320 Leu His Val Gly Val Met Arg Asn Arg Phe AspCys Ala Ile Val Leu 325 330 335 Leu Thr His Gly Ala Asn Ala Asp Ala ArgGly Glu His Gly Asn Thr 340 345 350 Pro Leu His Leu Ala Met Ser Lys AspAsn Val Glu Met Ile Lys Ala 355 360 365 Leu Ile Val Phe Gly Ala Glu ValAsp Thr Pro Asn Asp Phe Gly Glu 370 375 380 Thr Pro Thr Phe Leu Ala SerLys Ile Gly Lys Leu Gln Asp Leu Met 385 390 395 400 His Ile Ser Arg AlaArg Lys Pro Ala Phe Ile Leu Gly Ser Met Arg 405 410 415 Asp Glu Lys ArgThr His Asp His Leu Leu Cys Leu Asp Gly Gly Gly 420 425 430 Val Lys GlyLeu Ile Ile Ile Gln Leu Leu Ile Ala Ile Glu Lys Ala 435 440 445 Ser GlyVal Ala Thr Lys Asp Leu Phe Asp Trp Val Ala Gly Thr Ser 450 455 460 ThrGly Gly Ile Leu Ala Leu Ala Ile Leu His Ser Lys Ser Met Ala 465 470 475480 Tyr Met Arg Gly Met Tyr Phe Arg Met Lys Asp Glu Val Phe Arg Gly 485490 495 Ser Arg Pro Tyr Glu Ser Gly Pro Leu Glu Glu Phe Leu Lys Arg Glu500 505 510 Phe Gly Glu His Thr Lys Met Thr Asp Val Arg Lys Pro Lys ValMet 515 520 525 Leu Thr Gly Thr Leu Ser Asp Arg Gln Pro Ala Glu Leu HisLeu Phe 530 535 540 Arg Asn Tyr Asp Ala Pro Glu Thr Val Arg Glu Pro ArgPhe Asn Gln 545 550 555 560 Asn Val Asn Leu Arg Pro Pro Ala Gln Pro SerAsp Gln Leu Val Trp 565 570 575 Arg Ala Ala Arg Ser Ser Gly Ala Ala ProThr Tyr Phe Arg Pro Asn 580 585 590 Gly Arg Phe Leu Asp Gly Gly Leu LeuAla Asn Asn Pro Thr Leu Asp 595 600 605 Ala Met Thr Glu Ile His Glu TyrAsn Gln Asp Leu Ile Arg Lys Gly 610 615 620 Gln Ala Asn Lys Val Lys LysLeu Ser Ile Val Val Ser Leu Gly Thr 625 630 635 640 Gly Arg Ser Pro GlnVal Pro Val Thr Cys Val Asp Val Phe Arg Pro 645 650 655 Ser Asn Pro TrpGlu Leu Ala Lys Thr Val Phe Gly Ala Lys Glu Leu 660 665 670 Gly Lys MetVal Val Asp Cys Cys Thr Asp Pro Asp Gly Arg Pro 675 680 685 2112 basepairs nucleic acid double linear cDNA NO CDS 43..2106 22 GAATTCCGGGACGGTGGGGC CTCCCCACCT GCCCCGCAGA AG ATG CAG TTC TTT 54 Met Gln Phe Phe 1GGC CGC CTG GTC AAT ACC TTC AGT GGC GTC ACC AAC TTG TTC TCT AAC 102 GlyArg Leu Val Asn Thr Phe Ser Gly Val Thr Asn Leu Phe Ser Asn 5 10 15 20CCA TTC CGG GTG AAG GAG GTG GCT GTG GCC GAC TAC ACC TCG AGT GAC 150 ProPhe Arg Val Lys Glu Val Ala Val Ala Asp Tyr Thr Ser Ser Asp 25 30 35 CGAGTT CGG GAG GAA GGG CAG CTG ATT CTG TTC CAG AAC ACT CCC AAC 198 Arg ValArg Glu Glu Gly Gln Leu Ile Leu Phe Gln Asn Thr Pro Asn 40 45 50 CGC ACCTGG GAC TGC GTC CTG GTC AAC CCC AGG AAC TCA CAG AGT GGA 246 Arg Thr TrpAsp Cys Val Leu Val Asn Pro Arg Asn Ser Gln Ser Gly 55 60 65 TTC CGA CTCTTC CAG CTG GAG TTG GAG GCT GAC GCC CTA GTG AAT TTC 294 Phe Arg Leu PheGln Leu Glu Leu Glu Ala Asp Ala Leu Val Asn Phe 70 75 80 CAT CAG TAT TCTTCC CAG CTG CTA CCC TTC TAT GAG AGC TCC CCT CAG 342 His Gln Tyr Ser SerGln Leu Leu Pro Phe Tyr Glu Ser Ser Pro Gln 85 90 95 100 GTC CTG CAC ACTGAG GTC CTG CAG CAC CTG ACC GAC CTC ATC CGT AAC 390 Val Leu His Thr GluVal Leu Gln His Leu Thr Asp Leu Ile Arg Asn 105 110 115 CAC CCC AGC TGGTCA GTG GCC CAC CTG GCT GTG GAG CTA GGG ATC CGC 438 His Pro Ser Trp SerVal Ala His Leu Ala Val Glu Leu Gly Ile Arg 120 125 130 GAG TGC TTC CATCAC AGC CGT ATC ATC AGC TGT GCC AAT TGC GCG GAG 486 Glu Cys Phe His HisSer Arg Ile Ile Ser Cys Ala Asn Cys Ala Glu 135 140 145 AAC GAG GAG GGCTGC ACA CCC CTG CAC CTG GCC TGC CGC AAG GGT GAT 534 Asn Glu Glu Gly CysThr Pro Leu His Leu Ala Cys Arg Lys Gly Asp 150 155 160 GGG GAG ATC CTGGTG GAG CTG GTG CAG TAC TGC CAC ACT CAG ATG GAT 582 Gly Glu Ile Leu ValGlu Leu Val Gln Tyr Cys His Thr Gln Met Asp 165 170 175 180 GTC ACC GACTAC AAG GGA GAG ACC GTC TTC CAT TAT GCT GTC CAG GGT 630 Val Thr Asp TyrLys Gly Glu Thr Val Phe His Tyr Ala Val Gln Gly 185 190 195 GAC AAT TCTCAG GTG CTG CAG CTC CTT GGA AGG AAC GCA GTG GCT GGC 678 Asp Asn Ser GlnVal Leu Gln Leu Leu Gly Arg Asn Ala Val Ala Gly 200 205 210 CTG AAC CAGGTG AAT AAC CAA GGG CTG ACC CCG CTG CAC CTG GCC TGC 726 Leu Asn Gln ValAsn Asn Gln Gly Leu Thr Pro Leu His Leu Ala Cys 215 220 225 CAG CTG GGGAAG CAG GAG ATG GTC CGC GTG CTG CTG CTG TGC AAT GCT 774 Gln Leu Gly LysGln Glu Met Val Arg Val Leu Leu Leu Cys Asn Ala 230 235 240 CGG TGC AACATC ATG GGC CCC AAC GGC TAC CCC ATC CAC TCG GCC ATG 822 Arg Cys Asn IleMet Gly Pro Asn Gly Tyr Pro Ile His Ser Ala Met 245 250 255 260 AAG TTCTCT CAG AAG GGG TGT GCG GAG ATG ATC ATC AGC ATG GAC AGC 870 Lys Phe SerGln Lys Gly Cys Ala Glu Met Ile Ile Ser Met Asp Ser 265 270 275 AGC CAGATC CAC AGC AAA GAC CCC CGT TAC GGA GCC AGC CCC CTC CAC 918 Ser Gln IleHis Ser Lys Asp Pro Arg Tyr Gly Ala Ser Pro Leu His 280 285 290 TGG GCCAAG AAC GCA GAG ATG GCC CGC ATG CTG CTG AAA CGG GGC TGC 966 Trp Ala LysAsn Ala Glu Met Ala Arg Met Leu Leu Lys Arg Gly Cys 295 300 305 AAC GTGAAC AGC ACC AGC TCC GCG GGG AAC ACG GCC CTG CAC GTG GGG 1014 Asn Val AsnSer Thr Ser Ser Ala Gly Asn Thr Ala Leu His Val Gly 310 315 320 GTG ATGCGC AAC CGC TTC GAC TGT GCC ATA GTG CTG CTG ACC CAC GGG 1062 Val Met ArgAsn Arg Phe Asp Cys Ala Ile Val Leu Leu Thr His Gly 325 330 335 340 GCCAAC GCG GAT GCC CGC GGA GAG CAC GGC AAC ACC CCG CTG CAC CTG 1110 Ala AsnAla Asp Ala Arg Gly Glu His Gly Asn Thr Pro Leu His Leu 345 350 355 GCCATG TCG AAA GAC AAC GTG GAG ATG ATC AAG GCC CTC ATC GTG TTC 1158 Ala MetSer Lys Asp Asn Val Glu Met Ile Lys Ala Leu Ile Val Phe 360 365 370 GGAGCA GAA GTG GAC ACC CCG AAT GAC TTT GGG GAG ACT CCT ACA TTC 1206 Gly AlaGlu Val Asp Thr Pro Asn Asp Phe Gly Glu Thr Pro Thr Phe 375 380 385 CTAGCC TCC AAA ATC GGC AGA CAA CTA CAG GAT CTC ATG CAC ATC TCA 1254 Leu AlaSer Lys Ile Gly Arg Gln Leu Gln Asp Leu Met His Ile Ser 390 395 400 CGGGCC CGG AAG CCA GCG TTC ATC CTG GGC TCC ATG AGG GAC GAG AAG 1302 Arg AlaArg Lys Pro Ala Phe Ile Leu Gly Ser Met Arg Asp Glu Lys 405 410 415 420CGG ACC CAC GAC CAC CTG CTG TGC CTG GAT GGA GGA GGA GTG AAA GGC 1350 ArgThr His Asp His Leu Leu Cys Leu Asp Gly Gly Gly Val Lys Gly 425 430 435CTC ATC ATC ATC CAG CTC CTC ATC GCC ATC GAG AAG GCC TCG GGT GTG 1398 LeuIle Ile Ile Gln Leu Leu Ile Ala Ile Glu Lys Ala Ser Gly Val 440 445 450GCC ACC AAG GAC CTG TTT GAC TGG GTG GCG GGC ACC AGC ACT GGA GGC 1446 AlaThr Lys Asp Leu Phe Asp Trp Val Ala Gly Thr Ser Thr Gly Gly 455 460 465ATC CTG GCC CTG GCC ATT CTG CAC AGT AAG TCC ATG GCC TAC ATG CGC 1494 IleLeu Ala Leu Ala Ile Leu His Ser Lys Ser Met Ala Tyr Met Arg 470 475 480GGC ATG TAC TTT CGC ATG AAG GAT GAG GTG TTC CGG GGC TCC AGG CCC 1542 GlyMet Tyr Phe Arg Met Lys Asp Glu Val Phe Arg Gly Ser Arg Pro 485 490 495500 TAC GAG TCG GGG CCC CTG GAG GAG TTC CTG AAG CGG GAG TTT GGG GAG 1590Tyr Glu Ser Gly Pro Leu Glu Glu Phe Leu Lys Arg Glu Phe Gly Glu 505 510515 CAC ACC AAG ATG ACG GAC GTC AGG AAA CCC AAG GTG ATG CTG ACA GGG 1638His Thr Lys Met Thr Asp Val Arg Lys Pro Lys Val Met Leu Thr Gly 520 525530 ACA CTG TCT GAC CGG CAG CCG GCT GAA CTC CAC CTC TTC CGG AAC TAC 1686Thr Leu Ser Asp Arg Gln Pro Ala Glu Leu His Leu Phe Arg Asn Tyr 535 540545 GAT GCT CCA GAA ACT GTC CGG GAG CCT CGT TTC AAC CAG AAC GTT AAC 1734Asp Ala Pro Glu Thr Val Arg Glu Pro Arg Phe Asn Gln Asn Val Asn 550 555560 CTC AGG CCT CCA GCT CAG CCC TCA GAC CAG CTG GTG TGG CGG GCG GCC 1782Leu Arg Pro Pro Ala Gln Pro Ser Asp Gln Leu Val Trp Arg Ala Ala 565 570575 580 CGA AGC AGC GGG GCA GCT CCT ACT TAC TTC CGA CCC AAT GGG CGC TTC1830 Arg Ser Ser Gly Ala Ala Pro Thr Tyr Phe Arg Pro Asn Gly Arg Phe 585590 595 CTG GAC GGT GGG CTG TTG GCC AAC AAC CCC ACG CTG GAT GCC ATG ACC1878 Leu Asp Gly Gly Leu Leu Ala Asn Asn Pro Thr Leu Asp Ala Met Thr 600605 610 GAG ATC CAT GAG TAC AAT CAG GAC CTG ATC CGC AAG GGT CAG GCC AAC1926 Glu Ile His Glu Tyr Asn Gln Asp Leu Ile Arg Lys Gly Gln Ala Asn 615620 625 AAG GTG AAG AAA CTC TCC ATC GTT GTC TCC CTG GGG ACA GGG AGG TCC1974 Lys Val Lys Lys Leu Ser Ile Val Val Ser Leu Gly Thr Gly Arg Ser 630635 640 CCA CAA GTG CCT GTG ACC TGT GTG GAT GTC TTC CGT CCC AGC AAC CCC2022 Pro Gln Val Pro Val Thr Cys Val Asp Val Phe Arg Pro Ser Asn Pro 645650 655 660 TGG GAG CTG GCC AAG ACT GTT TTT GGG GCC AAG GAA CTG GGC AAGATG 2070 Trp Glu Leu Ala Lys Thr Val Phe Gly Ala Lys Glu Leu Gly Lys Met665 670 675 GTG GTG GAC TGT TGC ACG GAT CCA GAC GGG CGG CCG GAATTC 2112Val Val Asp Cys Cys Thr Asp Pro Asp Gly Arg Pro 680 685 688 amino acidsamino acid linear protein 23 Met Gln Phe Phe Gly Arg Leu Val Asn Thr PheSer Gly Val Thr Asn 1 5 10 15 Leu Phe Ser Asn Pro Phe Arg Val Lys GluVal Ala Val Ala Asp Tyr 20 25 30 Thr Ser Ser Asp Arg Val Arg Glu Glu GlyGln Leu Ile Leu Phe Gln 35 40 45 Asn Thr Pro Asn Arg Thr Trp Asp Cys ValLeu Val Asn Pro Arg Asn 50 55 60 Ser Gln Ser Gly Phe Arg Leu Phe Gln LeuGlu Leu Glu Ala Asp Ala 65 70 75 80 Leu Val Asn Phe His Gln Tyr Ser SerGln Leu Leu Pro Phe Tyr Glu 85 90 95 Ser Ser Pro Gln Val Leu His Thr GluVal Leu Gln His Leu Thr Asp 100 105 110 Leu Ile Arg Asn His Pro Ser TrpSer Val Ala His Leu Ala Val Glu 115 120 125 Leu Gly Ile Arg Glu Cys PheHis His Ser Arg Ile Ile Ser Cys Ala 130 135 140 Asn Cys Ala Glu Asn GluGlu Gly Cys Thr Pro Leu His Leu Ala Cys 145 150 155 160 Arg Lys Gly AspGly Glu Ile Leu Val Glu Leu Val Gln Tyr Cys His 165 170 175 Thr Gln MetAsp Val Thr Asp Tyr Lys Gly Glu Thr Val Phe His Tyr 180 185 190 Ala ValGln Gly Asp Asn Ser Gln Val Leu Gln Leu Leu Gly Arg Asn 195 200 205 AlaVal Ala Gly Leu Asn Gln Val Asn Asn Gln Gly Leu Thr Pro Leu 210 215 220His Leu Ala Cys Gln Leu Gly Lys Gln Glu Met Val Arg Val Leu Leu 225 230235 240 Leu Cys Asn Ala Arg Cys Asn Ile Met Gly Pro Asn Gly Tyr Pro Ile245 250 255 His Ser Ala Met Lys Phe Ser Gln Lys Gly Cys Ala Glu Met IleIle 260 265 270 Ser Met Asp Ser Ser Gln Ile His Ser Lys Asp Pro Arg TyrGly Ala 275 280 285 Ser Pro Leu His Trp Ala Lys Asn Ala Glu Met Ala ArgMet Leu Leu 290 295 300 Lys Arg Gly Cys Asn Val Asn Ser Thr Ser Ser AlaGly Asn Thr Ala 305 310 315 320 Leu His Val Gly Val Met Arg Asn Arg PheAsp Cys Ala Ile Val Leu 325 330 335 Leu Thr His Gly Ala Asn Ala Asp AlaArg Gly Glu His Gly Asn Thr 340 345 350 Pro Leu His Leu Ala Met Ser LysAsp Asn Val Glu Met Ile Lys Ala 355 360 365 Leu Ile Val Phe Gly Ala GluVal Asp Thr Pro Asn Asp Phe Gly Glu 370 375 380 Thr Pro Thr Phe Leu AlaSer Lys Ile Gly Arg Gln Leu Gln Asp Leu 385 390 395 400 Met His Ile SerArg Ala Arg Lys Pro Ala Phe Ile Leu Gly Ser Met 405 410 415 Arg Asp GluLys Arg Thr His Asp His Leu Leu Cys Leu Asp Gly Gly 420 425 430 Gly ValLys Gly Leu Ile Ile Ile Gln Leu Leu Ile Ala Ile Glu Lys 435 440 445 AlaSer Gly Val Ala Thr Lys Asp Leu Phe Asp Trp Val Ala Gly Thr 450 455 460Ser Thr Gly Gly Ile Leu Ala Leu Ala Ile Leu His Ser Lys Ser Met 465 470475 480 Ala Tyr Met Arg Gly Met Tyr Phe Arg Met Lys Asp Glu Val Phe Arg485 490 495 Gly Ser Arg Pro Tyr Glu Ser Gly Pro Leu Glu Glu Phe Leu LysArg 500 505 510 Glu Phe Gly Glu His Thr Lys Met Thr Asp Val Arg Lys ProLys Val 515 520 525 Met Leu Thr Gly Thr Leu Ser Asp Arg Gln Pro Ala GluLeu His Leu 530 535 540 Phe Arg Asn Tyr Asp Ala Pro Glu Thr Val Arg GluPro Arg Phe Asn 545 550 555 560 Gln Asn Val Asn Leu Arg Pro Pro Ala GlnPro Ser Asp Gln Leu Val 565 570 575 Trp Arg Ala Ala Arg Ser Ser Gly AlaAla Pro Thr Tyr Phe Arg Pro 580 585 590 Asn Gly Arg Phe Leu Asp Gly GlyLeu Leu Ala Asn Asn Pro Thr Leu 595 600 605 Asp Ala Met Thr Glu Ile HisGlu Tyr Asn Gln Asp Leu Ile Arg Lys 610 615 620 Gly Gln Ala Asn Lys ValLys Lys Leu Ser Ile Val Val Ser Leu Gly 625 630 635 640 Thr Gly Arg SerPro Gln Val Pro Val Thr Cys Val Asp Val Phe Arg 645 650 655 Pro Ser AsnPro Trp Glu Leu Ala Lys Thr Val Phe Gly Ala Lys Glu 660 665 670 Leu GlyLys Met Val Val Asp Cys Cys Thr Asp Pro Asp Gly Arg Pro 675 680 685 21bases nucleic acid single linear oligonucleotides NO 24 CATGGGACCCGCTGGCTTTC C 21 22 bases nucleic acid single linear oligonucleotides NO25 GGCAGGAACC GCCACTGGGG GC 22

What is claimed is:
 1. A composition comprising a purified phospholipaseenzyme characterized by (a) activity in the absence of calcium; (b) amolecular weight of 86 kD on SDS-PAGE; and (c) the presence of one ormore amino acid sequences selected from the group consisting ofYGASPLHXAK, MKDEVFR, EFGEHTK, VMLTGTLSDR, XXGAAPTYFRP and TVFGAK,wherein X represents any amino acid residue.
 2. The composition of claim1 wherein said enzyme is further characterized by activity in a mixedmicelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.3. The composition of claim 2 wherein said enzyme has a specificactivity of about 1 μmol to about 20 μmol per minute per milligram. 4.The composition of claim 1 wherein said enzyme is further characterizedby a pH optimum of
 6. 5. The composition of claim 1 wherein said enzymeis further characterized by the absence of stimulation by adenosinetriphosphate.
 6. An isolated polynucleotide comprising a nucleotidesequence selected from the group consisting of: (a) the nucleotidesequence of SEQ ID NO:16; (b) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:17; (c) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO:17 having activity in amixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (d) the nucleotidesequence of SEQ ID NO:18; (e) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:19; (f) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO:19 having activity in amixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (g) the nucleotidesequence of SEQ ID NO:20; (h) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:21; (i) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO:21 having activity in amixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (j) the nucleotidesequence of SEQ ID NO:22; (k) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:23; (l) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO:23 having activity in amixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (m) a nucleotidesequence capable of hybridizing with the sequence of any of (a)-(l)which encodes a peptide having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; and (n) allelicvariants of the sequence of (a), (d), (g) or (j).
 7. An expressionvector comprising the polynucleotide of claim 6 and an expressioncontrol sequence.
 8. A host cell transformed with the vector of claim 7.9. A process for producing a phospholipase enzyme, said processcomprising: (a) establishing a culture of the host cell of claim 8 in asuitable culture medium; and (b) isolating said enzyme from saidculture.
 10. A composition comprising a peptide made according to theprocess of claim
 9. 11. A composition comprising a peptide encoded bythe polynucleotide of claim
 6. 12. A composition comprising a peptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:17; (b) a fragment of the aminoacid sequence of SEQ ID NO:17 having activity in a mixed micelle assaywith 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (c) the aminoacid sequence of SEQ ID NO:19; (d) a fragment of the amino acid sequenceof SEQ ID NO:19 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (e) the amino acidsequence of SEQ ID NO:21; (f) a fragment of the amino acid sequence ofSEQ ID NO:21 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (g) the amino acidsequence of SEQ ID NO:23; and (h) a fragment of the amino acid sequenceof SEQ ID NO:23 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.
 13. A method foridentifying an inhibitor of phospholipase activity, said methodcomprising: (a) combining a phospholipid, a candidate inhibitorcompound, and a composition comprising a phospholipase enzyme peptide;and (b) observing whether said phospholipase enzyme peptide cleaves saidphospholipid and releases fatty acid thereby, wherein said compositionis the composition of claim
 1. 14. An inhibitor of phospholipaseactivity identified according to the method of claim
 13. 15. Apharmaceutical composition comprising a therapeutically effective amountof the inhibitor of claim 14 and a pharmaceutically acceptable carrier.16. A method of reducing inflammation comprising administering apharmaceutical composition of claim 15 to a mammalian subject.
 17. Acomposition comprising an antibody which binds to the peptide of thecomposition of claim
 1. 18. The composition of claim 17 wherein saidantibody is polyclonal.
 19. The composition of claim 17 wherein saidantibody is monoclonal.
 20. The polynucleotide of claim 6 comprising thenucleotide sequence of SEQ ID NO:16.
 21. The polynucleotide of claim 6comprising a nucleotide sequence encoding the amino acid sequence of SEQID NO:17.
 22. The polynucleotide of claim 6 comprising the nucleotidesequence of SEQ ID NO:18.
 23. The polynucleotide of claim 6 comprising anucleotide sequence encoding the amino acid sequence of SEQ ID NO:19.24. The polynucleotide of claim 6 comprising the nucleotide sequence ofSEQ ID NO:20.
 25. The polynucleotide of claim 6 comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO:21.
 26. Thepolynucleotide of claim 6 comprising the nucleotide sequence of SEQ IDNO:22.
 27. The polynucleotide of claim 6 comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO:23.
 28. Acomposition comprising a purified mammalian calcium independentphospholipase A₂/B enzyme.